Solid lipid particles, particles of bioactive agents and methods for the manufacture and use thereof

ABSTRACT

The present invention is in the area of administration forms and delivery systems for drugs, vaccines and other biologically active agents. More specifically the invention is related to the preparation of suspensions of colloidal solid lipid particles (SLPs) of predominantly anisometrical shape with the lipid matrix being in a stable polymorphic modification and of suspensions of micron and submicron particles of bioactive agents (PBAs); as well as to the use of such suspensions or the lyophilizates thereof as delivery systems primarily for the parenteral administration of preferably poorly water-soluble bioactive substances, particularly drugs, and to their use in cosmetic, food and agricultural products. 
     SLPs and PBAs are prepared by the following emulsification process: 
     (1) A solid lipid or bioactive agent or a mixture of solid lipids or bioactive agents is melted. 
     (2) Stabilizers are added either to the lipid or bioactive agent and to the aqueous phase or to the aqueous phase only depending on their physicochemical characteristics. 
     (3) Drugs or other bioactive substances to be incorporated into the SLPs may be melted together with the lipids if the physicochemical characteristics of the substance permit or may be dissolved, solubilized or dispersed in the lipid melt before homogenization. 
     (4) The aqueous phase is heated to the temperature of the melt before mixing and may contain for example stabilizers, isotonicity agents, buffering substances, cryoprotectants and/or preservatives. 
     (5) The molten lipid compounds and the bioactive agents are emulsified in an aqueous phase preferably by high-pressure homogenization.

RELATED APPLICATION

This application is a continuation-in-part of parent application Ser.No. 08/141,058, filed Oct. 26, 1993, now abandoned which in turn is acontinuation-in-part of application Ser. No. 08/027,501, filed Mar. 5,1993, now abandoned filed on Mar. 5, 1993 and the benefits of 35 USC 120are claimed relative to these two prior applications.

This invention relates to suspensions of particles of biodegradablelipids solid at room temperature, preferably triglycerides, which can beused as carriers for poorly water soluble drugs or other bioactiveagents, and to suspensions of particles constituted by biologicallyactive agents such as drugs, insecticides, fungicides, pesticides,herbicides and fertilizers, as well as to the lyophilizates thereof.Both systems can be prepared by a melt emulsification process.

The properties of the solid lipid particles (SLPs) includebiodegradability, avoidance of toxicologically active residues from theproduction process, enhanced physicochemical stability with regard tocoalescence and drug leakage, modified surface characteristics,controlled release of incorporated substances and modifiedbiodistribution. The particles can be prepared by an emulsificationprocess of molten material creating liquid droplets which formcrystalline anisometrical particles on cooling. The anisometricalparticles are of micron and submicron size, predominantly in the sizerange from 50 to 500 nm. The described suspensions have severaladvantages over other drug carrier systems deriving from the solidbiodegradable matrix being predominantly present in a β-polymorphicmodification (e g β', β₁, β₂), and not in an amorphous or α-crystallinestate.

The preparation of micron and submicron particles consisting of poorlywater-soluble bioactive agents (PBAS) by emulsification of the moltensubstance presents a novel process to reduce the particle size and/or tomodify the surface characteristics of powdered substances which can beaccomplished by inexpensive techniques and by the use of physiologicallyacceptable additives only. The suspensions of the particles are aneasy-to-handle product from the security point of view. The particles ofbioactive agents provide for the modified biodistribution andbioavailability of the formulated drugs or other bioactive substanceswhich implies a modification of the extent and rate of dissolution andabsorption, the circulation time, the site of action and the way ofdisposition of the drug or other bioactive substance. A reduction inparticle size below the micrometer range provides for the directintravenous administration of particles made from poorly water-solubledrugs without the need of a carrier vehicle.

FIELD OF THE INVENTION

The present invention is in the area of administration forms anddelivery systems for drugs, vaccines and other biologically activeagents such as insecticides, fungicides, pesticides, herbicides andfertilizers. More specifically, the invention is related to thepreparation of suspensions of colloidal solid lipid particles (SLPs)with the lipid matrix being in a stable polymorphic modification and ofsuspensions of micron and submicron particles of bioactive agents(PBAs), as well as to the use of such suspensions or the lyophilizatesthereof as delivery systems, primarily for the parenteral but also forthe peroral, nasal, pulmonary, rectal, dermal and buccal administrationof preferably poorly water-soluble bioactive substances, particularlydrugs; and to their use in cosmetic, food and agricultural products.These suspension systems provide for the controlled release ofincorporated or constituting substances as well as for the modifiedbiodistribution and bioavailability of incorporated or constitutingdrugs, which implies a modification of the extent and rate ofdissolution and absorption, the circulation time, the site of action andthe way of disposition of the drug.

BACKGROUND OF THE INVENTION

The parenteral, in particular the intravenous administration ofwater-insoluble or poorly water-soluble substances such as drugs orother biological materials often presents a problem to the formulator.Since the diameter of the smallest blood capillaries is only a fewmicrons the intravenous application of larger particles would lead tocapillary blockage. Solid drug substances are, however, commonlydisintegrated by milling and grinding, thereby generating particles froma few millimeters down to the micrometer size range which are too largeto be injected directly as an aqueous suspension. As a consequence,intravenous administration systems containing suspended particles ofwater-insoluble drugs are not commercially available due to the risk ofembolism. A further decrease in particle size is expensive, ineffectiveor even impossible by conventional techniques. Additionally, thereduction of solids to submicron-sized powders brings about heavydifficulties in handling these dry products such as an increased risk ofdust explosions and cross-contamination problems in a factoryenvironment. Moreover, such systems present a health risk for personsexposed to the possible inhalation and absorption of potent bioactivematerials. Up to now the only possibilities to administer poorlywater-soluble substances by the intravenous route are the use ofco-solvents or the development of carrier systems which incorporate suchsubstances in vehicles with hydrophilic surfaces.

Basic requirements of an ideal drug carrier system implybiodegradability, non-toxicity and non-immunogenicity. Moreover, thecarrier should be suitable for the intended route of administration, e gwith regard to particle size. Often a controlled release of theincorporated bioactive material is desired, for example when constantserum levels should be maintained over a long period of time or when thedrug exhibits only a low therapeutic index.

Furthermore, carrier systems can be employed to prolong the half-life ofcertain substances which are unstable due to rapid enzymatic orhydrolytic degradation in biological milieu. On the other hand theincorporation of drug in the carrier material also presents anopportunity to protect the host from the drug in case of non-selectivetoxic substances such as antitumour agents.

In many cases drug carrier systems are developed with the object todeliver drugs to site-specific targets under circumvention of uptake bythe reticuloendothelial system (RES). The rationale for such a drugtargeting is an enhancement of the drug's therapeutic efficacy by anincrease of the drug concentration at the target site with asimultaneous decrease at non-target sites, thereby rendering possible areduction of the administered dose. Thus, the toxicity of drugs, e.g.anticancer agents, can be diminished, leading to a decrease of sideeffects.

The prerequisite of a successful site-specific delivery implies acertain selectivity of the carrier system for the target tissue as wellas the accessibility of the desired target site. Targeting by theintravenous route of application is generally connected to an avoidanceor at least a reduction of carrier uptake by the RES except for thecases where a direct targeting to cells of the RES is desired. Clearanceof colloidal particles by the RES has been described to depend onparticle size as well as on particle surface characteristics such assurface charge and surface hydrophobicity. In general, small particlesare cleared less rapidly from the blood stream than large particleswhereas charged particles are taken up more rapidly than hydrophilicnon-charged particles. Due to these facts approaches to drug targetingare the modification of surface characteristics and the reduction ofparticle size.

Moreover, a small particle size is also required for the targeting ofdrugs to extravascular sites since extravasation is only feasiblethrough a receptor-mediated uptake by phagocytosis/pinocytosis or wherethe endothelial wall is fenestrated. These fenestrations can be foundfor example in the sinusoids of liver, spleen and bone-marrow and showdiameters of up to approximately 150 nm.

From the manufacturing point of view the ideal drug carrier systemshould be preparable without complications by easy-to-handle techniquesin a reproducible manner and possibly at low production costs. Theformulation should exhibit sufficient stability during preparation aswell as on storage.

In recent years several colloidal systems have received special interestfor their potential application as drug carriers, among them beingliposomes, lipid emulsions, microspheres and nanoparticles. However, allof the systems mentioned possess a certain number of draw-backs which sofar have prevented the break-through of any such system as a widespread,commercially exploited drug carrier.

Drug carrier systems in the micrometer size range are represented bymicrospheres consisting of a solid polymer matrix, and microcapsules inwhich a liquid or a solid phase is surrounded and encapsulated by apolymer film. Nanoparticles consist, like microspheres, of a solidpolymer matrix. Their mean particle size, however, lies in the nanometerrange. Both micro- and nanoparticles are generally prepared either byemulsion polymerization or by solvent evaporation techniques. Due tothese production methods micro- and nanoparticles bear the risk ofresidual contaminations from the production process like organicsolvents such as chlorinated hydrocarbons, as well as toxic monomers,surfactants and cross-linking agents, which may lead to toxicologicalproblems. Moreover, some polymeric materials such as polylactic acid andpolylactic-glycolic acid degrade very slowly in vivo so that multipleadministration could lead to polymer accumulation associated withadverse side effects. Other polymers such as polyalkylcyanoacrylatesrelease toxic formaldehyde on degradation in the body.

Drug carrier systems for parenteral administration based on lipids areliposomes and submicron lipid emulsions. Although such systems consistof physiological components only, thus reducing toxicological problemsthere is a number of disadvantages associated with these lipid carriers.

Liposomes are spherical colloidal structures in which an internalaqueous phase is surrounded by one or more phospholipid bilayers. Thepotential use of liposomes as drug delivery systems has been disclosedinter alia in the U.S. Pat. No. 3,993,754 (issued Nov. 23, 1976 toRahmann and Cerny), U.S. Pat. No. 4,235,871 (issued Nov. 25, 1980 toPapahadjopoulos and Szoka) and U.S. Pat. No. 4,356,167 (issued Oct. 26,1982 to L. Kelly). The major drawbacks of conventional liposomes aretheir instability on storage, the low reproducibility of manufacture,the low entrapment efficiency and the leakage of drugs.

According to the IUPAC definition, in an emulsion liquids or liquidcrystals are dispersed in a liquid. Lipid emulsions for parenteraladministration consist inter alia of liquid oil droplets, predominantlyin the submicron size range, dispersed in an aqueous phase andstabilized by an interfacial film of emulsifiers. Typical formulationsare disclosed in the Jap. Pat. No 55,476/79 issued May 7, 1979 toOkamota, Tsuda and Yokoyama. The preparation of a drug containing lipidemulsion is described in WO 91/02517 issued Mar. 7, 1991 to Davis andWashington. The susceptibility of these lipid emulsions towards theincorporation of drugs is relatively high due to the mobility of drugmolecules within the internal oil phase since diffusing molecules caneasily protrude into the emulsifier film causing instabilities whichlead to coalescence. Furthermore, release of incorporated drugs fromlipid emulsions is relatively fast so that the possibilities for asustained drug release are limited.

Fountain et al (U.S. Pat. No. 4,610,868 issued Sep. 9, 1986) developedlipid matrix carriers which are described as globular structures of ahydrophobic compound and an amphiphatic compound with diameters fromabout 500 nm to about 100,000 nm. The hydrophobic compound can be liquidor solid. The preparation techniques, however, employ organic solventsand are thus associated with the problem of complete solvent removal.

So-called lipospheres disclosed by Domb et al (U.S. patent applicationSer. No. 435,546 lodged Nov. 13, 1989 and now abandoned; Int. Appl. NoPCT/US90/06519 filed Nov. 8, 1990) are described as suspensions ofsolid, water-insoluble microspheres made of a solid hydrophobic coresurrounded by a phospholipid layer. Lipospheres are claimed to providefor the sustained release of entrapped substances controlled by thephospholipid layer. They can be prepared by a melt or by a solventtechnique, the latter creating toxicological problems if the solvent isnot completely removed.

A slow release composition of fat or wax and a biologically activeprotein, peptide or polypeptide suitable for parenteral administrationto animals is disclosed in U.S. patent application Ser. No. 895,608lodged Aug. 11, 1986, and now abandoned to Staber, Fishbein and Cady(EP-A-0 257 368). The systems are prepared by spray drying and consistof spherical particles in the micrometer size range up to 1.000 micronsso that intravenous administration is not possible.

Problems with the formulation of water-insoluble or poorly water-solublesubstances are not restricted to the parenteral route of administration.Thus, the peroral bioavailability of drugs is related to theirsolubility in the gastrointestinal tract (GIT), and it is generallyfound that poorly water-soluble drugs exhibit a low bioavailability.Moreover, the dissolution of drugs in the GIT is influenced by theirwettability. Substances with apolar surfaces are scarcely wetted inmedia so that their dissolution rate is very slow.

In an attempt to improve the intestinal absorption of lipophilic drugs,Eldem et al (Pharm. Res. 8, 1991, 47-54) prepared lipid micropellets byspray-drying and spray-congealing processes. The micropellets aredescribed as spherical particles with smooth surfaces. The lipids are,however, present in unstable polymorphic forms, and polymorphic phasetransitions occur during storage so that the product properties areconstantly changing (T. Eldem et al, Pharm. Res. 8, 1991, 178-184).Thus, constant product qualities cannot be assured.

Lipid nanopellets for peroral administration of poorly bioavailabledrugs are disclosed in EP 0 167 825 of Aug. 8, 1990 to P. Speiser. Thenanopellets represent drug-loaded fat particles solid at roomtemperature and small enough to be persorbed. Persorption is thetransport of intact particles through the intestinal mucosa into thelymph and blood compartment. The lipid nanopellets are prepared byemulsifying molten lipids in an aqueous phase by high-speed stirring.After cooling to room temperature the pellets are dispersed bysonication.

OBJECT OF THE INVENTION

Considering the limitations of conventional drug carriers such asliposomes, lipid emulsions, nanoparticles and microspheres as outlinedabove there is an obvious demand for a carrier system for the controlleddelivery of poorly water-soluble bioactive substances to circumvent thedrawbacks of traditional systems particularly with regard topreparation, stability, toxicity and modification of biodistribution.

The present invention introduces a new type of carrier systemcharacterized as non-spherically shaped particles composed ofcrystalline lipids, preferably triglycerides, and physiologicallyacceptable additives as well as a process for the manufacturing thereof.These carriers provide for the controlled delivery of poorlywater-soluble substances such as drugs or other biological materialsprimarily by the parenteral but also by the peroral, nasal, pulmonary,rectal, dermal and buccal route of administration, and will hereinafterbe referred to as solid lipid particles (SLPs).

SLPs are characterized as lipidic particles of a solid physical state inthe micro- and predominantly in the nanometer size range. The shape ofthe particles is mainly anisometric which is a consequence of the matrixforming lipids present in a β-polymorphic modification (e g β', β₁ β₂),and not in an amorphous or α-crystalline state. The properties of theSLPs include: (1) biodegradability and non-toxicity; (2) the ability toincorporate poorly water-soluble substances; (3) improved chemical andphysical stability; (4) the possibility to prepare a dry storageformulation; (5) control of release characteristics of incorporatedsubstances; and (6) modified surface characteristics. As a result ofthese properties SLPs overcome many of the problems encountered withconventional drug carrier systems.

The present invention is supposed to bring about the followingadvantages as derived from the characteristics of the SLPs describedabove:

(1) SLPs can be prepared of biodegradable, pharmacologically acceptablecompounds only and are, therefore, non-toxic. Additionally, thepreparation of SLPs avoids the employment of organic solvents or anyother potentially toxic additives, thus evading the contamination of theproduct with residual impurities.

(2) SLPs possess an enhanced chemical stability as compared withconventional lipid emulsions based on liquid triglyceride oils owing tothe lower degree of unsaturated fatty acids of solid triglycerides.Moreover, SLPs exhibit a better physical stability due to the solidnature of the lipid matrix which is expectedly more resistant tocoalescence than fluid emulsion droplets. Furthermore, the lipid matrixis present in a stable β-polymorphic modification (e g β', β₁, β₂).Thus, the product properties will not change significantly duringlong-term storage due to polymorphic transformations.

(3) Suspensions of SLPs can be lyophilized by freeze-drying to provide awater-free storage system that exhibits a good long-term stability. Thelyophilized powder can be redispersed in water, buffer or solutions ofamino acids, carbohydrates and other infusion solutions directly beforeuse or can be processed into other pharmaceutical formulations.

(4) Due to their lipophilic nature SLPs are suited for thesolubilization of lipophilic and poorly water-soluble substances byentrapment into the lipid matrix. Compared to lipid emulsions SLPs aresupposed to be less sensitive to the incorporation of drugs or otherbioactive materials due to their solid nature. Drugs or other bioactivematerials diffusing into the emulsifier film or recrystallizing close tothe surface perturb the stabilizing film of emulsion droplets,increasing the risk of film rupture followed by coalescence. Incontrast, film elasticity and film viscosity are of minor importance inthe case of solid suspension particles since they cannot coalescebecause of the rigid nature of the lipid.

(5) Drug release from the lipid carrier can be controlled for example bythe composition of the lipid matrix by the choice of stabilizing agentsas well as by the size of SLPs. Drug leakage is hindered by the solidstate of the carrier due to the restricted drug diffusion.

(6) Drugs or bioactive substances exhibiting a short half-life due toenzymatic or hydrolytic degradation can be protected from rapiddecomposition by incorporation within the lipid carrier since thehydrophobic matrix prevents the access of water to the incorporated drugon storage as well as in body fluids.

(7) The incorporation into SLPs of drugs or other bioactive substanceswith a low bioavailability due to poor solubility in thegastrointestinal tract (GIT) can enhance the bioavailability of suchsubstances because these are solubilized in the biodegradable lipidmatrix and are thus present in the dissolved state.

(8) Due to the anisometrical shape of SLPs the specific surface area islarger than that of spherical particles of the same volume. Substanceswith a low peroral bioavailability can be absorbed faster and to ahigher degree in the GIT when they are incorporated in anisometricalSLPs than in spherical lipid particles of the same volume, due to thelarger surface area of SLPs since the potential, site of action forlipolytic enzymes is larger.

(9) The surface characteristics of SLPs can be modified by variation ofthe lipid composition, use of different stabilizers, exchange ofsurfactants and/or adsorption of polymeric compounds. The modificationof surface characteristics brings about the possibility to modify the invivo distribution of the carrier and the incorporated substance. In caseof intravenous administration this implies a modified uptake by the RESwith the potential for drug targeting.

(10) Due to the submicron particle size of SLPs there is no risk ofembolism by parenteral administration. Since SLPs can be prepared downto a particle size of about 50 nm, they possess the opportunity ofextravasation through fenestrations of the endothelial wall. Thereby,drugs can be targeted to extravascular sites such as the bone marrow,for example.

Furthermore, the present invention introduces a new type of deliverysystem for the parenteral, peroral, nasal, pulmonary, rectal, dermal andbuccal administration of drugs or other bioactive substances as well asthe process for the manufacturing thereof. These formulations aresuspensions of particles formed by bioactive substances with modifiedsurface characteristics and/or a reduced particle size as compared tothe powdered substance, and will hereinafter be referred to as particlesof bioactive agents (PBAs). The preparation of PBAs can avoid theemployment of any toxicologically active additives such as organicsolvents or toxic monomers, and can be accomplished by easy-to-handletechniques.

PBAs can be used in the following fields of application:

a) as a parenteral delivery system with modified biodistribution forsparingly water-soluble bioactive substances without the need of acarrier vehicle;

b) as a delivery system according to a) for peroral, nasal, pulmonary,rectal, dermal and buccal administration;

c) as a formulation for the peroral administration of drugs with a poorbioavailability due to a low dissolution rate in the gastrointestinaltract;

d) as a delivery system for use in agricultural applications;

e) the lyophilizate of formulations a) to d) as a reconstitutable powderwith an enhanced stability on storage.

Owing to the special characteristics of the present invention PBAs aresupposed to bring about the following advantages over conventionalpharmaceutical delivery systems:

1) The formulation of poorly water-soluble drugs or other bioactivesubstances as micron and submicron particles avoids the need of acarrier system for their parenteral application, thereby circumventingthe disadvantages of conventional drug carriers like liposomes, lipidemulsions, nanoparticles and microspheres.

2) PBAs can be prepared by easy-to-handle techniques in a reproducibleway. There are no problems to foresee for the scaling up of themanufacturing process.

3) Since the particles consist of the pure bioactive compound with onlysmall amounts of stabilizers the drug-load capacity of the drugparticles is high.

4) The release of drugs or other bioactive compounds from theformulation can be controlled by the choice of amphiphatic compoundsemployed to stabilize the particles.

5) The preparation of PBAs can avoid the use of toxicologically activeadditives.

6) A water-free storage system with enhanced stability can be produced,for example by freeze-drying of the PBA dispersions.

7) The surface characteristics of PBAs can be modified by the choice ofamphiphatic compounds used as stabilizers as well as by the attachmentof so-called homing devices for the targeting of drugs, for examplemonoclonal antibodies or carbohydrate moieties. The surfacemodifications give rise to a modified bioavailability andbiodistribution with regard to the extent and rate of absorption, thecirculation time, the site of action and the way of disposition of thebioactive substance. The modification of surface characteristics alsoprovides the opportunity to avoid or at least to reduce the uptake ofintravenously administered particles by cells of the RES.

8) Since the particles can be prepared with a size below 100 nm to 200nm they possess the opportunity for extravasation by fenestrations ofthe endothelial wall. Thereby, drugs can be targeted to extravascularsites such as the bone-marrow, for example.

9) A reduction in particle size to the nanometer size range generallynot achievable by milling or grinding leads to an enormous increase ofthe specific surface area of the particles. Since the peroralbioavailability of drugs or other bioactive substances is related to thespecific surface area via the dissolution rate of the substance in thegastrointestinal tract the submicron sized particles give rise to anenhanced bioavailability of drugs poorly soluble in the GIT.

10) Hydrophobic substances can be formulated as PBAs with hydrophilicsurfaces. Hydrophilic surfaces provide for a good wettability of theparticles, for example in the GIT, facilitating dissolution of thecompound. Thus, the bioavailability can be increased.

11) The process of manufacturing of PBAs involves inexpensiveeasy-to-handle techniques only and provides a product which is safe tohandle. Since the particles are present in a liquid dispersion there isno risk of dust explosions, cross-contamination or inhalation ofbioactive substances as often encountered with the production ofextremely fine powders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Transmission electron micrograph of tripalmitate SLPs of Example1 after 5 months of storage at room temperature. The bar represents 400nm.

FIG. 2 Differential scanning calorimetric (DSC) thermogram of a) puretripalmitate and of b) tripalmitate SLPs of Example 1. The transitionpeaks correspond to the melting of the crystalline polymorph.

FIG. 3 Synchrotron radiation wide angle X-ray diffraction pattern oftripalmitate SLPs of Example 2. The reflexions correspond to thecrystalline polymorph.

FIG. 4 Transmission electron micrograph of unstable hard fat SLPs ofExample 5. The SLP dispersion gelatinized on storage by forming athree-dimensional network. The bar corresponds to 1000 nm.

FIG. 5 Particle size distribution of tripalmitate SLPs of Example 1after 15 months of storage. The graph represents the result of amultiangle PCS measurement.

FIG. 6 PCS particle size distribution of a 10% tripalmitate SLPdispersion compared to that of the commercial lipid emulsion Intralipid®10%.

FIG. 7 Influence of microfluidization time on the mean particle size ofhard fat SLPs of Example 3.

FIG. 8 Stability on storage of hard fat SLPs of Example 3 as indicatedby the development of the mean particle size with storage time(monitored period: 12 months).

FIG. 9 Influence of homogenization pressure on the mean particle size oftripalmitate SLPs.

FIG. 10 Influence of the number of homogenization passes on the meanparticle size of tripalmitate SLPs (no 0 corresponds to the crudedispersion prepared by sonication).

FIG. 11 Influence of sonication time on the mean particle size oftripalmitate SLPs prepared by probe sonication.

FIG. 12 Influence of type and amount of emulsifier on the mean particlesize of tripalmitate SLPs prepared according to Example 13.

FIG. 13 Effect of bile salts as co-emulsifier on the mean particle sizeof different phospholipid stabilized SLP dispersions of Example 14.

FIG. 14 Particle size distribution of trimyristate SLPs of Example 15 asdetermined by laser diffractometry.

FIG. 15 The physical state of different drug-loaded SLPs a) at 20° C.and b) at 38° C. determined by synchrotron radiation wide-angle X-raydiffraction.

FIG. 16 Dissolution speed of ibuprofen PBAs of Example 37.

FIG. 17 Polarized microscopic picture of lidocaine PBAs of Example 38(magnification: 150×).

FIG. 18 Polarized microscopic picture of lidocaine raw material employedfor the production of lidocaine PBAs (magnification: 150×).

DESCRIPTION OF THE INVENTION

The present invention relates to suspensions of micron and submicronparticles of biodegradable lipids solid at room temperature (solid lipidparticles, SLPs), to suspensions of particles of meltable bioactivesubstances (PBAs), to lyophilizates thereof and to methods for themanufacturing thereof.

Solid lipid particles (SLPs) are of predominantly anisometrical shapewhich is a consequence of the lipid matrix being present in aβ-polymorphic modification (e g β', β₁, β₂) or in a polymorphic stateanalogous to that of β-crystals of triglycerides and not in an amorphousor α-crystalline-like state. SLPs can be used as carrier systemsprimarily for the parenteral but also for the peroral, nasal, pulmonary,rectal, dermal and buccal administration of poorly water-solublesubstances such as drugs or other biologically active materials. Theapplication of SLPs is, however, not restricted to the administration ofpharmaceuticals to humans or animals. SLPs can also be used in cosmetic,food and agricultural products. SLPs are novel lipid structures withproperties that overcome many of the problems associated with previouslydescribed carrier systems.

The matrix of SLPs is constituted by biocompatible hydrophobic materialswhich are solid at room temperature and have melting points ranging fromapproximately 30° to 120° C. The preferred matrix constituents are solidlipids (fats) such as mono-, di- and triglycerides of long-chain fattyacids; hydrogenated vegetable oils; fatty acids and-their esters; fattyalcohols and their esters and ethers; natural or synthetic waxes such asbeeswax and carnauba wax; wax alcohols and their esters, sterols such ascholesterol and its esters, hard paraffins, as well as mixtures thereof.The carrier material must be compatible with the agent to beincorporated.

Lipids are known to exhibit a pronounced polymorphism. This can bedefined as the ability to reveal different unit cell structures incrystal, originating from a variety of molecular conformations andmolecular packings. Depending on the conditions, glycerides, forexample, may crystallize in three different polymorphic forms termedalpha (α), beta prime (β') and beta (β) according to the classificationof Larsson (K. Larsson, 1966, Acta Chem. Scand. 20, 2255-2260). Thesepolymorphic modifications characterized by a particular carbon chainpacking may differ significantly in their properties such as solubility,melting point and thermal stability. Transformations take place from αto β' to β, the transition being monotropic. The β-form is thethermodynamically most stable polymorph, whereas α is the least stableand will transform more or less rapidly into the more stable polymorphsβ' and β, depending on the thermal conditions. This transformation isaccompanied by a change of physicochemical properties.

In the described suspensions of SLPs the lipid matrix is predominantlypresent in a stable polymorphic modification. Although on cooling, thedispersed melt metastable polymorphs such as the α-form may occurintermediately a stable polymorph is formed within several hours or daysafter preparation of the dispersions.

The suspensions of SLPs can be stabilized by amphiphatic compounds suchas ionic and non-ionic surfactants. Suitable stabilizers include but arenot limited to the following examples: naturally occurring as well assynthetic phospholipids, their hydrogenated derivatives and mixturesthereof, sphingolipids and glycosphingolipids; physiological bile saltssuch as sodium cholate, sodium dehydrocholate, sodium deoxycholate,sodium glycocholate and sodium taurocholate; saturated and unsaturatedfatty acids or fatty alcohols; ethoxylated fatty acids or fatty alcoholsand their esters and ethers; alkylaryl-polyether alcohols such astyloxapol; esters and ethers of sugars or sugar alcohols with fattyacids or fatty alcohols; acetylated or ethoxylated mono- anddiglycerides; synthetic biodegradable polymers like block co-polymers ofpolyoxyethylene and polyoxypropyleneoxide; ethoxylated sorbitanesters orsorbitanethers; amino acids, polypeptides and proteins such as gelatineand albumin; or a combination of two or more of the above mentioned.

The aqueous phase in which the SLPs are dispersed can containwater-soluble or dispersable stabilizers; isotonicity agents such asglycerol or xylitol; cryoprotectants such as sucrose, glucose, trehaloseetc; electrolytes; buffers; antiflocculants such as sodium citrate,sodium pyrophosphate or sodium dodecylsulfate; preservatives.

Depending on the characteristics of the employed stabilizers thecoexistence of other colloidal structures such as micelles and vesiclesin suspensions of SLPs cannot be ruled out.

Substances particularly suitable for the entrapment into SLPs are drugsor other bioactive compounds which are poorly water-soluble, show a lowbioavailability, are badly absorbed from the intestinum, and/or will berapidly degraded in biological environment by chemical or enzymaticalprocesses, as well as low-specific active substances which are highlytoxic at non-target sites. In case it is desired to incorporate arelatively water-soluble compound into SLPs it is necessary to decreasethe water-solubility of this compound, which can be achieved for exampleby using a water-insoluble derivative of the compound such as an acid orbase, a complex, or a lipophilic precursor.

Drugs or bioactive agents particularly suited for incorporation intoSLPs are antibiotics such as fosfomycin, fosmidomycin and rifapentin;antihypertensives such as minoxidil, dihydroergotoxine and endralazine;antihypotensives such as dihydroergotamine; systemic antimycotics suchas ketoconazole and griseofulvin; antiphlogistics such as indomethacin,diclofenac, ibuprofen, ketoprofen and pirprofen; antiviral agents suchas aciclovir, vidarabin and immunoglobulines; ACE inhibitors such ascaptopril and enalapril; betablockers such as propranolol, atenolol,metoprolol, pindolol, oxprenolol and labetalol; bronchodilators such asipratropiumbromide and sobrerol; calcium antagonists such as diltiazem,flunarizin, verapamil, nifedipin, nimodipin and nitrendipin; cardiacglycosides such as digitoxin, digoxin, methyidigoxin and acetyidigoxin;cephalosporins such as ceftizoxim, cefalexin, cefalotin and cefotaxim;cytostatics such as chlormethin, cyclophosphamid, chlorambucil,cytarabin, vincristin, mitomycin C, doxorubicin, bleomycin, cisplatin,taxol, penclomedine and estramustin; hypnotics such as flurazepam,nitrazepam and lorazepam; psychotropic drugs such as oxazepam, diazepamand bromazepam; steroid hormones such as cortisone, hydrocortisone,prednisone, prednisolone, dexamethasone, progesterone, pregnanolone,testosterone and testosteroneundecanoat; vasodilators such asmolsidomin, hydralazin and dihydralazin; cerebral vasodilators such asdihydroergotoxin, ciclonicat and vincamin; lipophilic vitamins such asvitamins A, D, E, K and their derivates.

The bioactive substances can be located in the core of SLPs where theyare dissolved, solubilized or dispersed in the matrix, and/or in thestabilizer layer(s) surrounding the particle matrix, and/or can beadsorbed to the surface of SLPs. The bioactive substances can bedissolved or crystalline or amorphous or a mixture of thesecrystallographic states.

SLPs can be prepared by an emulsification process which exhibits certainsimilarities to the preparation of lipid(oil)-in-water emulsions but ismainly characterized by its basic differences as will be outlined below.The process is described as follows:

(1) The solid lipid or the mixture of lipids is melted.

(2) The stabilizers are added either to the lipid and to the dispersionmedium or to the dispersion medium only, depending on theirphysicochemical characteristics. The choice of stabilizers and theadmixture regime are not comparable with those applied for lipid(oil)-in-water emulsions, which is evident from the below examples.Stabilizers may also be added or exchanged after homogenization, forexample by adsorption of polymers or by dialysis of water-solublesurfactants.

(3) Drugs or other bioactive substances to be incorporated into the SLPsmay be melted together with the lipids if the physicochemicalcharacteristics of the substance permit, or may be dissolved,solubilized or dispersed in the lipid melt before homogenization.

(4) The dispersion medium is heated to the temperature of the meltbefore mixing and may contain for example stabilizers, isotonicityagents, buffering substances, cryoprotectants and/or preservatives.

(5) The melted lipid compounds are emulsified in the dispersion medium,preferably by high pressure homogenization, but emulsification is alsopossible by sonication, high speed stirring, vortexing and vigorous handshaking. The way of homogenization determines the particle size of theSLPs.

The basic differences to the preparation of lipid-in-water emulsionsbeside the choice and admixture regime of the stabilizers are related tothe following steps:

(6) After homogenization the dispersion can be sterilized by standardtechniques such as autoclaving or filtration through a 0.2 μm sterilefilter provided the particles are small enough not to be retained by thefilter. These steps have to be performed before the system is cooledbelow the recrystallization temperature. Moreover, contaminations whichcould lead to heterogenous nucleation should be avoided. It is thereforeadvisable to remove particulate contaminations from the dispersions byfiltration prior to cooling below the recrystallization temperature. Thepore size of the filter should be chosen sufficiently large so as not toretain the lipid particles.

(7) The dispersions are allowed to stand to cool off at room temperatureforming SLPs by recrystallization of the dispersed lipids. Duringcooling the dispersion may be agitated by a magnetic stirrer forexample.

(8) In a subsequent step the dispersion medium is reduced in volume forexample by evaporation or it can be removed by standard techniques suchas filtration, ultrafiltration or freeze-drying, thus yielding awater-free storage system which can be reconstituted prior to use. Thelyophilized powder can also be processed into other pharmaceutical,cosmetic, food or agricultural formulations such as powders, tablets,capsules etc.

SLPs are typically solid particles of anisometrical shape asdemonstrated by FIG. 1 which shows a transmission electron micrograph ofa freeze-fractured specimen of the SLPs of Example 1. The anisometricalparticle shape results from crystallization of the lipid matrix in theβ-polymorphic form. Solidification of the amorphous fat orcrystallization of the unstable α-polymorph generally reveals sphericalparticles. The presence of the stable β-form could be detected bydifferential scanning calorimetry (FIG. 2) and synchrotron radiationwide-angle X-ray diffraction (FIG. 3).

The particle size of SLPs depends on the type and amount of emulsifierand on the emulsification technique and conditions (see below). Theresolidification of the molten lipids prior to homogenization should beavoided because size reduction by homogenization is substantiallyimpeded if the particles are solid during this step. To ensure that themolten lipids do not solidify prior to homogenization, i e beforesmaller particles can be formed, the dispersion medium is heated toapproximately the same temperature as the melt before the two phases aremixed so that the melt will not be cooled down by the addition of thedispersion medium.

SLPs in the nanometer size range are obtained by high-pressurehomogenization. The particles show a relatively narrow particle sizedistribution with mean particle sizes by number of approximately 50-300nm as determined by photon correlation spectroscopy (PCS). Thedispersions of SLPs are stable on storage for more than 18 months. Thus,the long-term stability is similar to that of submicron o/w emulsionsused for parenteral nutrition. Long-term stability data of other solidlipid-based carrier dispersions described in the patent literature suchas lipospheres (A. Domb et al, Int. Appl. No PCT/US90106519 filed Nov.8, 1990) and lipid nanopellets (P. Speiser, Eur. Pat. No 0167825 issuedAug. 8, 1990) could not be found. Domb describes phospholipid stabilizedtristearate lipospheres with a seven day stability as "exceptionallystable".

It turned out that the stabilization of SLP suspensions requires thepresence of a highly mobile stabilizing agent in the dispersion mediumin such a way that the amount of highly mobile stabilizers in thedispersion medium is, after emulsification, sufficient to stabilizenewly created surfaces during recrystallization (see below). Bile salts,especially in combination with nonelectrolytic compounds such asglycerol in concentrations used to achieve blood isotony, have proved tobe very efficient in this respect. SLPs stabilized by phospholipidsalone, or in combination with nonelectrolytic compounds such as glycerolin concentrations used to achieve blood isotony, tend to form semi-solidointment-like gels as shown on the transmission electron micrograph ofFIG. 4, whereas the addition of sodium glycocholate to the aqueous phaseprevents this gel formation (B. Siekmann and K. Westesen, 1992, Pharm.Pharmacol. Lett. 1, 123-126).

A molar phospholipid to bile salt ratio between 2:1 and 4:1 turned outto be most effective regarding the initial stabilization duringhomogenization as well as the long-term storage stability of SLPdispersions. These phospholipid/bile salt ratios are above the ratio offormation of mixed micelles and coincide with a swollen lamellar phaseof mixed lecithin/bile salt layers in the ternary phase diagram of thesystem lecithin/bile salt/water. Data therefore suggest thatstabilization is most effective if the bile salt is not bound to mixedmicelles and that during stabilization of SLPs the bile salt moleculesare inserted in the phospholipid layer on the surface of the particles.

SLPs can also be sterically stabilized by nonionic surfactants. Stericstabilization of SLPs requires, however, a relatively high amount ofsurfactants with lipid/surfactant ratios up to 1:1. It can be observedin general that the stability of SLPs decreases with increasinglipid/surfactant ratio.

The amount of emulsifier required for surface stabilization of thedispersed particles is higher than in conventional lipid emulsions, forexample such as used in parenteral nutrition. This effect can beattributed to the crystallization of the molten lipids afterhomogenization. Since the lipids typically do not recrystallize or exist(on storage) in the form of ideal spheres but as anisometric particlesthere is a large increase in surface area as compared to the droplets ofthe emulsified molten lipids or of conventional lipid emulsions,respectively. The additional surfaces newly generated duringrecrystallization or polymorphic transitions of the dispersed lipidsneed to be stabilized immediately on formation to avoid particleaggregation. Therefore, the preparation of stable SLP dispersionsrequires the presence of a reservoir of stabilizing agents afteremulsification.

The choice of stabilizers cannot be deduced from compositions andstabilization mechanisms for oil-in-water emulsions but is dependent onthe existence of highly mobile stabilizers due to the formationmechanism of the anisometrical particles. In colloid and surface science"highly mobile" generally refers to free diffusion in the dispersionmedium at a high diffusion velocity. With regard to the stabilization ofcolloidal solid lipid particles the diffusion velocity should besufficiently high to reach freshly created particle surfaces (especiallyduring re-crystallization of the lipid) before particle aggregation cantake place, in order to exert a stabilizing action at the lipid/waterinterface to prevent particle aggregation. Sufficiently high diffusionalvelocities are typically observed with substances which do not form aseparate phase in the dispersion medium according to the phase rule setup by Gibbs. Highly mobile stabilizers can be of ionic or nonionicnature. Typically, these stabilizers dissolve molecularly in thedispersion medium and/or form micelles. Micelles are known to be highlydynamic structures characterized by a fast exchange of molecules betweenmicellar aggregates and the dispersion medium. The monomers in thedispersion medium are immediately available for surface stabilization.In contrast, stabilizing agents that tend to form a separate phase inthe dispersion medium are not sufficiently mobile to stabilize freshlycreated surfaces before particle aggregation can take place. Thesestabilizers are therefore not suitable as sole stabilizers of SLPdispersions. Phospholipids are an example of stabilizing agents thatform a separate phase in the dispersion medium. It is well known thatphospholipids form closed lamellar structures, so called vesicles, inaqueous media, and that the exchange rate of phospholipid moleculesbetween vesicles and the aqueous phase is extremely small compared tothat of micelles. Phospholipid molecules are therefore bound invesicular structures and are not immediately available to stabilizenewly created surfaces during recrystallization of the lipid particles.Consequently, phospholipids alone, although suitable as effectivestabilizers of lipid emulsions, cannot efficiently stabilize SLPsuspensions as is evident from Examples 5 and 13. In fact, preparingSLPs with a standard composition of lipid emulsions, for example 10% fatand 1.2% phospholipids, results in unstable SLP dispersions. Even higherconcentrations of phospholipids such as 20% or 60% lecithin related tothe fat phase are not sufficient to stabilize SLP dispersions asdemonstrated in Example 5.

Although stated in the patent literature (e g Domb et al in U.S. Pat.No. 435,546 issued Nov. 13, 1989, and now abandoned, and Int. Appl. NoPCT/US90/0651 9 filed Nov. 8, 1990), fine suspensions of solid lipidsare not equivalent to submicron lipid emulsions in that respect that theinner phase is only replaced by solid fats instead of liquid ones. Thephysicochemical properties of lipid suspensions such as SLPs differsubstantially from that of lipid emulsions. As a consequence of thesedifferences lipid suspensions cannot be prepared and treated analogouslyto lipid emulsions. One basic difference of SLPs is their much largerparticle surface area as outlined above so that SLPs require a higheramount of surfactants which additionally need to be highly mobile toimmediately stabilize the new surfaces created when the molten lipidrecrystallizes or transforms into the stable β-polymorph. The secondbasic difference is that once SLPs are formed by recrystallization ofthe molten lipid any renewed melting of the small particles may resultin an instability of the dispersions if there is no excess of mobilestabilizer in the aqueous phase which is not adsorbed to particlesurfaces where it is immobilized. A further requirement forphysicochemical stability of SLPs in contrast to oil-in-water emulsionsis the absence of particulate impurities which could promoteheterogenous nucleation. It is therefore advisable to remove particulatecontaminations from the dispersions by filtration prior to cooling belowthe recrystallization temperature. Moreover, non-electrolytic compoundsused to achieve blood isotony such as glycerol turned out to promote thestability of SLP dispersions.

The present invention also relates to suspensions of particles ofbioactive agents (PBAs). Sparingly water-soluble substances such asdrugs, insectcides, fungicides, pesticides, herbicides, fertilizers,nutrients, cosmetics etc which are meltable in the temperature rangefrom approximately 30° to 120° C. can be formulated as PBAs by aprocedure similar to the preparation of SLPs as described above. Thematrix of PBAs is constituted by the bioactive agent itself.

PBAs present a novel type of delivery system and can be characterized aspredominantly submicron and/or micron particles of bioactive agentssuspended in an aqueous media stabilized by amphiphatic compounds. PBAspossess modified surface characteristics which can be controlled by thechoice of amphiphiles and/or a reduced particle size of the matrixconstituting compound as compared to the powdered substance. Thesecharacteristics give rise to a modified biodistribution andbioavailability of the formulated drugs or other bioactive substanceswhich implies a modification of the extent and rate of dissolution andabsorption, the circulation time, the site of action and the way ofdisposition of the drug or other bioactive substance. Thephysicochemical properties of PBAs depend strongly on thecharacteristics of the bioactive agent of which they are formulated, onthe type and amount of stabilizing agents as well as on the way ofemulsification. Suspensions and lyophilizates of PBAs can be used forthe peroral, nasal, pulmonary, rectal, dermal, buccal and, depending onthe particle size, also for the parenteral administration of poorlywater-soluble drugs or other biologically active compounds. Moreover,PBAs can also be employed in cosmetic, food and agricultural products,in particular for the formulation of poorly water-soluble herbicides andpesticides.

The matrix of PBAs is constituted by practically insoluble or sparinglywater-soluble agents with melting points preferably below 100° C. or themelting points of which can be decreased to below 100° C. by theaddition of certain adjuvants. Substances particularly suitable for theformulation as PBAs are drugs or other bioactive materials which arepoorly water-soluble, show a low bioavailability and/or are badlyabsorbed from the intestinum. Examples of such substances comprise butare not limited to:

Anesthetics and narcotics such as butanilicaine, fomocaine,isobutambene, lidocaine, risocaine, prilocaine, pseudococaine,tetracaine, trimecaine, tropacocaine and etomidate; anticholinergicssuch as metixen and profenamine; antidepressives, psychostimulants andneuroleptics such as alimenazine, binedaline, perazine, chlorpromazine,fenpentadiol, fenanisol, fluanisol, mebenazine, methylphenidate,thioridazine, toloxaton and trimipramine; antiepileptics such asdimethadion and nicethamide; antimycotics such as butoconazole,chlorphenesin, etisazole, exalamid, pecilocine and miconazole;antiphlogistics such as butibufen and ibuprofen; bronchodilators such asbamifylline; cardiovascular drugs such as alprenolol, butobendine,cloridazole, hexobendine, nicofibrate, penbutolol, pirmenol,prenylamine, procaine amide, propatylnitrate, suloctidil, toliprolol,xibendol and viquidile; cytostatics such as asperline, chlorambucile,chlornaphhazine, mitotane, estramustine, taxol, penclomedine andtrofosfamide; hyperemic drugs such as capsaicine and methylnicotinate;lipid reducers such as nicoclonate, oxprenolol, pirifibrate, simfibrateand thiadenol; spasmolytics such as aminopromazine, caronerine,difemerine, fencarbamide, tiropramide and moxaverine; testosteronederivates such as testosterone enantate andtestosterone-(4-methylpentanoate); tranquilizers such as azaperone andburamate; virustatics such as arildon; vitamin A derivates such asretinol, retinol acetate and retinol palmitate; vitamin E derivates suchas tocopherol acetate, tocopherol succinate and tocopherol nicotinate;menadione; cholecalciferol; insecticides, pesticides and herbicides suchas acephate, cyfluthrin, azinphosphomethyl, cypermethrine, substitutedphenyl thiophosphates, fenclophos, permethrine, piperonal, tetramethrineand trifluraline.

As with SLPs, suspensions of PBAs can be stabilized by amphiphaticcompounds. Principally the same ionic and nonionic surfactants which maybe employed for the stabilization of SLPs are also suitable for thepreparation of PBA suspensions. The choice of stabilizing agents dependson the physicochemical properties of both the bioactive substance andthe dispersion medium as well as on the desired surface characteristicsof the particles.

The aqueous phase in which the PBAs are dispersed should containwater-soluble (or dispersable) stabilizers; isotonicity agents such asglycerol or xylitol; cryoprotectants such as sucrose, glucose, trehaloseetc; electrolytes; buffers; antiflocculants such as sodium citrate,sodium pyrophosphate or sodium dodecylsulfate; preservatives. Althoughwater is the preferred dispersion medium the invention is, however, notrestricted to aqueous dispersions alone but can be extended to any otherpharmacologically acceptable liquid such as ethanol, propylene glycoland methyl-isobutyl-ketone, or a mixture thereof.

Depending on the characteristics of the employed stabilizers thecoexistence of other colloidal structures such as micelles and vesiclesin suspensions of PBAs cannot be ruled out.

Suspensions of PBAs are typically prepared by an emulsification processsimilar to that of SLPs. The molten drug or bioactive substance or amixture of such compounds is emulsified in a pharmacologicallyacceptable liquid immiscible with the melt, preferably by high-pressurehomogenization. Emulsification is also possible by sonication,high-speed stirring, vortexing and vigorous hand shaking. The liquid isheated to the temperature of the melt before mixing and may contain forexample isotonicity agents, buffering substances, cryoprotectants and/orpreservatives.

The stabilizing amphiphatic compounds are added either to the melt andto the liquid or to the liquid only, depending on their physicochemicalcharacteristics. Stabilizers may also be added or exchanged afterhomogenization, for example by the adsorption of polymers or bydialysis.

The PBAs manufactured according to the above described process can becategorized in two distinguishable groups.

The PBAs of the first group are characterized in that they arewater-insoluble at the temperature of emulsion preparation and will notbe solubilized by the excess of stabilizers or form micelles bythemselves, the particle size of PBAs remaining unchanged before andafter cooling to room temperature.

The PBAs of the second group are characterized in that they are partlywater-soluble at the temperature of emulsion preparation and/or are ableto form mixed micelles by the excess of stabilizers and/or form micellesby themselves, leading to an increase of particle size after cooling toroom temperature due to, for example, crystal growth and/orprecipitation of dissolved bioactive agent from the supersaturatedsolution and/or due to mass transport from smaller to larger particles,for example in micelles and/or by processes such as Ostwald ripening.

In a subsequent step the liquid phase can be removed by freeze-drying,for example, producing a reconstitutable powder which can also beprocessed into other pharmaceutical formulations.

PBAs are finely dispersed particles consisting of a matrix of bioactivematerial surrounded by one or more layers rich in surfactant. Theparticle size and the size distribution as well as the particle shapeand the surfactant coating depend on the properties and amounts of thematrix forming bioactive substances and the stabilizing agents, theratio of bioactive material to amphiphatic compounds as well as on theway of emulsification.

EXAMPLES Example 1

Method of preparation of tripalmitate SLPs.

In a thermostatized vial 4.0 g tripalmitate (tripalmitin, 99% pure,Fluka) is heated to 75° C. to melt the lipid. In the lipid melt 0.48 gsoy bean lecithin (Lipoid S 100, Lipoid KG) is dispersed by probesonication (MSE Soniprep 150) until the dispersion appears opticallyclear. 0.16 g sodium glycocholate (glycocholic acid, sodium salt 99%,Sigma) and 4 mg thiomersal (Synopharm) is dissolved in 36 ml bidistilledwater. The aqueous phase is heated to 75° C. and added to the lipidmelt. A crude dispersion is produced by probe sonication forapproximately 2 minutes. The crude dispersion is transferred to athermostatized high-pressure homogenizer (APV Gaulin Micron Lab 40) andpassed 5 times through the homogenizer at a pressure of 500 bar.Homogenization with this equipment is accomplished by extrusion througha small ring-shaped orifice. The homogenized dispersion is allowed tostand at room temperature to cool off. The dispersion reveals traceamounts of visible fat particles which are separated from the dispersionby filtering it through a 0.45 μm sterile filter.

The importance of the admixture of highly mobile surfactants such asbile salts with regard to the particle size distribution and thestability of SLP dispersions is demonstrated below, e g in Examples 2, 5to 7 and 13 to 15.

The mean particle size after preparation (by number) of the tripalmitateSLPs determined by photon correlation spectroscopy (PCS, MalvernZetasizer 3) is 205 nm. After 15 months of storage the particles show novisible signs of aggregation, creaming, sedimentation or phaseseparation. A PCS multiangle measurement (Malvern Zetasizer 3, detectionat five different angles: 50, 70, 90, 110 and 130 degrees) reveals amonomodal particle size distribution (by number) with a peak at 250 nm(FIG. 5).

At temperatures below the melting point of the lipid matrix tripalmitateSLPs are predominantly anisometrical particles as demonstrated in FIG. 1which is a transmission electron micrograph of a freeze-fracturedspecimen of the tripalmitate SLPs of Example 1. Before preparation ofthe specimen the sample is stored at room temperature for 5 months. Thesample is freeze-fractured at 173 K in a freeze-fracture unit BAF 400(Balzers AG, CH-Liechtenstein). Fast freezing is accomplished by slushinto melting propane. Shadowing of the sample is performed withplatinum/carbon (layer thickness 2 nm) at 45 degrees and with purecarbon at 90 degrees for replica preparation. Replica are cleaned with a1:1 (v/v) chloroform/ethanol mixture. Replica on uncoated grids areviewed with an electron microscope EM 300 (Philips, D-Kassel).

In the anisometrical tripalmitate particles the glyceride is present inthe stable β-crystalline polymorph as indicated by thermoanalyticalinvestigations. FIG. 2 presents a differential scanning calorimetric(DSC) thermogram of SLPs of Example 1 and of pure tripalmitate. Thesamples are weighed accurately into standard aluminium pans. Thermogramsare recorded from 20° C. to 90° C. at a scan rate of 10° C./minute on aPerkin Elmer calorimeter DSC-7. The detected transition peaks correspondto the melting of tripalmitate β-crystals. The melting point oftripalmitate SLPs is shifted to a lower temperature compared to that ofpure tripalmitate due to the presence of phospholipids and due to thesmall crystallite size.

Example 2

Method of preparation of isotonic tripalmitate SLPs.

7.0 g tripalmitate (tripalmitin, Fluka) is melted in a vialthermostatized at 75° C. 840 mg soy bean lecithin (Lipoid S 100, LipoidKG) is dispersed in the tripalmitate melt as described in Example 1. Theaqueous phase containing 1.575 g glycerol, 280 mg sodium glycocholateand 4 mg thiomersal is heated to 75° C. and added to the lipid melt to aweight of 70 g. A crude dispersion is produced by sonication forapproximately 2 minutes. The crude dispersion is transferred to athermostatized high-pressure homogenizer (APV Gaulin Micron Lab 40) andpassed 10 times through the homogenizer at a pressure of 800 bar. Thehomogenized dispersion is allowed to stand at room temperature to cooloff.

The mean particle size by number of the isotonic tripalmitate SLPsdetermined by PCS is 125.9 nm after preparation and 116.2 nm after 50days of storage, i.e. there was practically no particle growth. Theslight deviations of the values fall into the range of accuracy of thesizing method. The PCS particle size distribution is compared to that ofthe commercially available lipid emulsion for parenteral nutritionIntralipid™ in FIG. 6. Intralipid™ is composed of 10% soy bean oil, 1.2%fractionated phospholipids and 2.25% glycerol dispersed in water forinjection. It can be observed that the particle size distribution oftripalmitate SLPs of Example 2 is significantly smaller and more narrowthan that of Intralipid™. In contrast to Example 1 the addition ofglycerol results in a noticeable difference in the particle sizedistribution. Whereas the SLP dispersion of Example 1 contains traceamounts of visible suspension particles after being cooled to roomtemperature from the hot emulsion no macroscopically visible suspensionparticles are observed in the dispersion of Example 2.

Investigations by synchrotron radiation wide-angle X-ray diffraction(FIG. 3) and differential scanning calorimetry reveal that thetripalmitate in SLPs is present in the stable β-polymorphic form at roomtemperature.

Example 3

Preparation of hard fat SLPs by microfluidization.

3.0 g hard fat (Witepsol™ W35, Huls AG) is melted in a thermostatizedvial at 75° C. 1.8 g soy bean lecithin (Phospholipon 100, Natterman) isdispersed in the tripalmitate melt as described in Example 1. Theaqueous phase containing 375 mg sodium glycocholate, 2.25 g glycerol and10 mg thiomersal is heated to 75° C. and added to the lipid melt to aweight of 100 g. A crude dispersion is produced by ultra-turraxvortexing for approximately 2 minutes. The crude dispersion istransferred to a microfluidizer (Microfluidics Microfluidizer M-11OT), ahigh-pressure homogenizer of the jet-stream principle which is immersedin a thermostatized water bath (70° C.). The dispersion is cycledthrough the microfluidizer for 10 minutes and allowed to stand at roomtemperature to cool off.

The mean particle size of hard fat SLPs after preparation is 45.9 nm asdetermined by PCS.

During homogenization a sample for particle sizing is drawn each minutein order to monitor the time course of homogenization. FIG. 7 displaysthe mean particle size versus homogenization time. The mean particlediameter is decreasing with time and levels off at the end ofhomogenization.

Example 4

Long-term stability of hard fat SLPs prepared by microfluidization.

The stability of hard fat SLPs is monitored over a period of one year.During this time the sample is stored in a refrigerator at approximately+4° C. After certain time intervals the particle size distribution ofthe sample is determined by PCS. FIG. 8 demonstrates that the meanparticle size of hard fat SLPs is practically constant over themonitored period of one year.

Example 5

Preparation of unstable SLPs dispersions.

In a thermostatized vial 4.0 g tripalmitate (Dynasan 116, Huls AG) isheated to 75° C. to melt the lipid. In the lipid melt 0.48 g soy beanlecithin (Lipoid S 100, Lipoid KG) is dispersed by probe sonication (MSESoniprep 150) until the dispersion appears optically clear. 4 mgthiomersal and 0.9 g glycerol is dissolved in 35.6 ml bidistilled water.The aqueous phase is heated to 75° C. and added to the lipid melt. Acrude dispersion is produced by probe sonication for approximately 2minutes. The crude dispersion is transferred to a thermostatizedhigh-pressure homogenizer (APV Gaulin Micron Lab 40) and passed 5 timesthrough the homogenizer at a pressure of 500 bar. The homogenizeddispersion is allowed to stand at room temperature to cool off. Onstorage the SLP dispersion becomes a milky semi-solid, ointment-likegel.

3.0 g hard fat (Witepsol™ W35, Huls AG) is melted in a thermostatizedvial at 75° C. 1.8 g soy bean lecithin (Phospholipon 100, Natterman) isdispersed in the tripalmitate melt as described in Example 1. Theaqueous phase containing 10 mg thiomersal is heated to 75° C. and addedto the lipid melt to a weight of 100 g. A crude dispersion is producedby ultra-turrax vortexing for approximately 2 minutes. The crudedispersion is transferred to a microfluidizer (MicrofluidicsMicrofluidizer M-11OT) which is immersed in a thermostatized water bath(70° C.). The dispersion is cycled through the microfluidizer for 10minutes and allowed to stand at room temperature to cool off. On storagethe SLP dispersion becomes a turbid semi-solid, ointment-like gel. Atransmission electron micrograph of this gel is presented in FIG. 4.

In a thermostatized vial 4.0 g tripalmitate (Dynasan 116, Huls AG) isheated to 80° C. to melt the lipid. In the lipid melt 0.8 g of a soybean lecithin mixture (Lipoid S 75, Lipoid KG) is dispersed by probesonication (MSE Soniprep 150) until the dispersion appears opticallyclear. 4 mg thiomersal is dissolved in 35.6 ml bidistilled water. Theaqueous phase is heated to 80° C. and added to the lipid melt. A crudedispersion is produced by probe sonication for approximately 2 minutes.The crude dispersion is transferred to a thermostatized high pressurehomogenizer (APV Gaulin Micron Lab 40) and passed 5 times through thehomogenizer at a pressure of 800 bar. The homogenized dispersion isfilled in a glass vial and allowed to stand at room temperature to cooloff. On cooling to room temperature the dispersion forms semi-solidlooking fat aggregates on the wall of the glass vial. The dispersiongelatinizes when shear forces are applied, for example by passing itthrough a hypodermic syringe.

Obviously the use of phospholipids only as stabilizers, as found incommercial parenteral oil-in-water emulsions, does not yield stablesystems in the case of SLP suspensions. Even the employment ofphospholipids such as Lipoid S 75 which induces a considerably highnegative net charge cannot provide a sufficient stabilization.Electrostatic repulsion alone cannot be the basic stabilizationmechanism of SLPs as will be further outlined in Examples 6, 7 and 13.

Example 6

Preparation of tripalmitate SLPs sterically stabilized by tyloxapol.

A series of tripalmitate SLP dispersions stabilized by tyloxapol(Eastman Kodak) are prepared with varying lipid/surfactant ratios. TheSLP dispersions are manufactured according to the following procedure:

Tyloxapol is dissolved in heated bidistilled water while the temperatureis held below the cloud point of tyloxapol (approximately 90°-95° C.).The tyloxapol solution of a temperature of 80° C. is added to the moltentripalmitate or, respectively, tripalmitate/lecithin dispersion of thesame temperature. A crude emulsion is prepared by probe sonication forapproximately 2 minutes. Then the crude emulsion is passed 5 timesthrough a high-pressure homogenizer at a pressure of 1200 bar. Thehomogenized dispersion is allowed to stand at room temperature to cooloff. All dispersions contain 2.5% glycerol and 0.01 % thiomersal.

Table 1 gives the composition of the prepared SLP dispersions and theirmean particle size after preparation (by number) as determined by PCS.The asterix (*) in the particle size column indicates that thedispersions display a bimodal size distribution with particle sizesconsiderably larger than indicated by the mean particle size. It turnsout that sterically stabilized SLP dispersions require a high amount ofsurfactant in order to obtain homogenously sized SLPs. In case of SLPsstabilized by tyloxapol and phospholipids the ratio of the surfactantsneeds to be optimized In the present series a tyloxapol/lecithin ratioof at least 1:1 turned out to yield homogenously sized SLPs. Withincreasing ratio the mean particle size is decreasing. As with examples1 to 3 the addition of a highly mobile surfactant which is able to formmicelles is required to obtain stable dispersions. The high amount ofsurfactant is needed to create a reservoir of surfactant in thedispersion medium that can provide enough surfactant molecules at themoment when the molten lipids recrystallize and form anisometricalparticles with a large specific surface area.

                  TABLE 1                                                         ______________________________________                                        SLP dispersions sterically stabilized by tyloxapol.                           Composition (w %)                                                             TP    Tyl         PL      Mean particle size (nm)                             ______________________________________                                        10    2           --      138.0*                                              10    4           --      84.9                                                10    0.7         2       487.4*                                              10    1           2       207.4*                                              10    2           2       102.8                                               10    4.5         3       60.9                                                ______________________________________                                         Abbreviations:                                                                TP = tripalmitate, Tyl = tyloxapol, PL = phospholipids (Lipoid S 100).   

Example 7

Preparation of tripalmitate SLPs sterically stabilized by poloxamers.

1.2 g soy bean lecithin (Lipoid S 100, Lipoid KG) is dispersed in 4.0 gmolten tripalmitate (Dynasan 116, Huls AG) by probe sonication at atemperature of 80° C. 1.8 g poloxamer (Pluronic™ F68, BASF), 0.9 gglycerol and 4 mg thiomersal is dissolved in 32.1 g bidistilled waterheated to 80° C. The heated solution is added to the lipid melt and acrude dispersion is prepared by 2 minutes probe sonication. The crudedispersion is transferred to a thermostatized high-pressure homogenizer(APV Gaulin Micron Lab 40) and passed 5 times through the homogenizer ata pressure of 1200 bar. The homogenized dispersion is allowed to standat room temperature to cool off.

The poloxamer stabilized SLPs display a monomodal size distribution witha mean particle size (by number) after preparation of 77.9 nm determinedby PCS. Due to the presence of an excess of highly mobile surfactant inthe aqueous phase the system is stabilized on recrystallization of themolten lipids and a gelation as found with systems stabilized byphospholipids only does not occur.

Example 8

The influence of homogenization pressure on the mean particle size ofSLPs.

SLPs of the following composition are prepared at differenthomogenization pressures. The SLP dispersions are composed of 3%tripalmitate (Dynasan 116, Huls AG), 1.5% tyloxapol, 1% soy beanlecithin (Lipoid S 100, Lipoid KG), 0.01% thiomersal and bidistilledwater to 100% (by weight). The lecithin is dispersed in the moltentripalmitate (80° C.) by probe sonication until the dispersion appearsoptically clear. Tyloxapol is dissolved in warm water (80° C.)containing thiomersal. The SLP dispersions are prepared as described inExample 6.

FIG. 9 displays the influence of homogenization pressure on the meanparticle size of the SLPs. With increasing pressure the particle size isdecreasing and the particle size distribution becomes more narrow.

Example 9

The influence of homogenization passes on the mean particle size ofSLPs.

Tripalmitate SLPs composed of 3% tripalmitate (Dynasan 116, Huls AG),1.5% tyloxapol, 1% soy bean lecithin (Lipoid S 100, Lipoid KG), 0.01%thiomersal and bidistilled water to 100% (by weight) is prepared at apressure of 800 bar as described in Example 6. Samples for sizemeasurements are taken from the dispersion after preparation of thecrude emulsion and after each pass through the homogenizer.

FIG. 10 presents the influence of the number of homogenization passes onthe mean particle size of SLPs which is decreasing with increasingnumber of passes.

Example 10

Preparation of SLPs by probe sonication--Influence of sonication time onthe mean particle size of SLPs.

In a sonication vial thermostatized at 80° C. 1.20 g tripalmitate ismelted. In the lipid melt 0.40 g soy bean lecithin (Lipoid S 100) isdispersed by probe sonication until the dispersion appears opticallyclear. 0.60 g tyloxapol and 4 mg thiomersal is dissolved in bidistilledwater heated to 80° C. The aqueous phase is added to the lipid melt andan SLP dispersion is prepared by probe sonication at 80° C. Thesonicator operates at 50% of its maximum power. At certain timeintervals (1, 5, 10 and 15 minutes) samples are taken from thedispersion for size measurements. After 30 minutes probe sonication isstopped and the dispersion is allowed to stand at room temperature tocool off.

The influence of sonication time on the mean particle size of the SLPsis displayed in FIG. 11. With increasing sonication time the meanparticle size is decreasing and the size distribution becomes morenarrow.

Example 11

Preparation of SLPs by stirring.

An SLP dispersion composed as in Examples 9 and 10 is prepared by use ofa heated magnetic stirrer (Pyro-Magnestir, Lab-Line). The lecithin isdispersed in the tripalmitate as described before. The heated aqueousphase is added to the melt. A dispersion is produced by stirring themixture for 30 minutes at a temperature of 80° C. The dispersion isallowed to stand at room temperature to cool off.

The mean particle size after preparation (by volume) of the SLPdispersion is 59.5 μm determined by laser diffractometry (MalvernMastersizer MS20). The maximum particle size measured is 250 μm. Incontrast to high pressure homogenization and probe sonication, stirringproduces relatively large particles in the micrometer size range.

Example 12

Influence of the matrix constituent on the mean particle size of SLPs.

SLP dispersions composed of 10% matrix constituent, 1.2% soy beanlecithin (Lipoid S 100), 0.4% sodium glycocholate, 2.25% glycerol and0.01 % thiomersal in bidistilled water to 100% are prepared as describedin Example 1. Five different matrix constituents are employed: the waxescetylpalmitate and white bees-wax and the triglycerides trilaurate,trimyristate and tripalmitate.

Table 2 presents the PCS mean particle sizes of the different SLPdispersions and the melting points of the matrix constituents.

                  TABLE 2                                                         ______________________________________                                        Influence of matrix constituents.                                             Matrix     Melting point (°C.)                                                                 Mean size of SLPs (nm)                                ______________________________________                                        Cetylpalmitate                                                                           45.5         141.0                                                 White bees-wax                                                                           62.5         195.3                                                 Trilaurate 45.0         137.2                                                 Trimyristate                                                                             56.5         161.1                                                 Tripalmitate                                                                             63.0         209.2                                                 ______________________________________                                    

The mean particle size of SLPs is increasing with the melting point ofthe matrix constituent.

Example 13

Influence of emulsifier type and amount on the mean particle size andstability of SLPs.

Tripalmitate SLP dispersions with different types and amounts ofemulsifiers are prepared as described in Example 2. The composition ofthe different batches is given in Table 3. All dispersions contain 2.25%glycerol and 0.01% thiomersal.

The mean particle size of the different batches of SLPs is presented inFIG. 12. The mean particle size depends on the type and amount ofemulsifier.

                  TABLE 3                                                         ______________________________________                                        Compositions of SLP batches (in w %).                                         Batch no TP     PL           SGC  Plu                                         ______________________________________                                        1        10%    1.2%         --   --                                          2        10%    1.2%         0.4% --                                          3        10%    2.4%         0.4% --                                          4        10%    --           --   1.8%                                        5        10%    --           --   3.6%                                        ______________________________________                                         Abbreviations:                                                                TP = tripalmitate, PL = phospholipids (Lipoid S 100), SGC = sodium            glycocholate, Plu = Pluronic F68.                                        

The combination of phospholipids and bile salts is most efficient withregard to the mean particle size and the stability. The systemstabilized by phospholipids only gelatinizes and forms an ointment-likesemi-solid gel on storage. The systems stabilized by Pluronic F68 tendto gelatinize when shear forces are applied, i e when the particles areforced to get closer to each other. Obviously the steric stabilizationby poloxamers is not sufficient in this case. As a result the optimumstabilization is that by a surfactant combination of emulsifiers thatare present in and act from the lipid side (such as phospholipids) andof emulsifiers that constitute a reservoir of highly mobile surfactantmolecules in the dispersion medium (such as bile salts, tyloxapol andpoloxamers). Though repulsion forces represent an important factor forthe long-term stability, the basic mechanism of SLP stabilization is thehigh mobility of the excess of surfactant which provides for theimmediate surface coverage of newly created surfaces duringrecrystallization of the molten lipids.

Example 14

Effect of bile salt as co-emulsifier of phospholipid stabilized SLPs.

Phospholipid-stabilized SLP dispersions employing different matrices(tripalmitate, hard fat) are prepared with or without the addition ofbile salt (sodium glycocholate) to the aqueous phase according to themethod described in Example 1. All dispersions contain 2.25% glyceroland 0.01% thiomersal. Emulsification of the crude dispersions isperformed by high pressure homogenization (APV Gaulin Micron Lab 40)under different homogenization conditions. The following dispersionswere prepared:

    ______________________________________                                        Composition          Homogenization conditions                                ______________________________________                                        7.0 g TP, 0.84 g PL, 62.2 g H.sub.2 O                                                              3 × 500 bar                                        7.0 g TP, 0.84 g PL, 0.28 g BS, 61,9 g H.sub.2 O                                                   3 × 500 bar                                        7.0 g TP, 0.84 g PL, 62.2 g H.sub.2 O                                                              10 × 1200 bar                                      7.0 g TP, 0.84 g PL, 0.28 g BS, 61.9 g H.sub.2 O                                                   10 × 1200 bar                                      7.0 g HF, 0.84 g PL, 62.2 g H.sub.2 O                                                              3 × 500 bar                                        7.0 g HF, 0.84 g PL, 0.28 g BS, 61.9 g H.sub.2 O                                                   3 × 500 bar                                        7.0 g HF, 0.84 g PL, 62.2 g H.sub.2 O                                                              10 × 1200 bar                                      7.0 g HF, 0.84 g PL, 0.28 g BS, 61.9 g H.sub.2 O                                                   10 × 1200 bar                                      ______________________________________                                         Abbreviations:                                                                BS = bile salt; H.sub.2 O = bidistilled water, HF = hard fat (Witepsol        W35); PL = phospholipids (Lipoid S 100); TP = tripalmitate.              

The mean particle size of the dispersions as determined by PCS afterpreparation is presented in FIG. 13. This example demonstrates theeffect of bile salts as co-emulsifier in the aqueous phase on theparticle size of phospholipid stabilized SLPs. It is clearly shown thatthe addition of bile salts reduces the mean particle size of SLPs by upto 57%. Thus, by the use of bile salts as co-emulsifier extremely finedispersions can be obtained. The effect of the bile salt can beattributed to the high mobility of this micelle forming ionic surfactantwhich enables the surfactant molecules to immediately cover freshlygenerated surfaces during the homogenization process. The phospholipidswhich tend to form liquid crystalline structures in the aqueous phaseare not sufficiently mobile to provide the immediate stabilization offreshly created particles so that instantaneous coalescence occurs incase there is no highly mobile co-surfactant in the aqueous phase.

Example 15

Preparation of trimyristate SLPs stabilized by a lecithin/bile saltblend.

In a thermostatized vial 7.0 g trimyristate (Dynasan 114, Huls AG) ismelted at 70° C. 0.96 g phospholipids (Lipoid S 100) are dispersed inthe melt by probe sonication. A solution of 280 mg sodium glycocholate,1.6 g glycerol and 7 mg thiomersal in 61 ml bidistilled water is heatedto 70° C. and added to the melt. A crude dispersion is prepared by probesonication for approximately 2 minutes. The crude dispersion istransferred to a high pressure homogenizer (APV Gaulin Micron Lab 40)thermostatized at approximately 90° C. and is passed 5 times through thehomogenizer at a pressure of 500 bar. The homogenized dispersion isallowed to stand at room temperature to cool off.

The mean particle size after preparation determined by PCS is 155.7 nm.In laser diffractometry (Malvern Mastersizer MS20) no particles above 1μm can be detected. The particle size distribution derived from laserdiffractometry is presented in FIG. 14. This example demonstrates thatthe use of bile salts as co-emulsifier of phospholipid stabilized SLPsefficiently prevents the formation of particles larger than 1 μm due tothe rapid coverage of freshly created surfaces during homogenization,thereby minimizing immediate coalescence.

Example 16

Preparation of tripalmitate SLPs without Ultrasonication (method A).

In a thermostatized vial 4.0 g tripalmitate (Dynasan 116, Huls AG) ismelted at 85° C. 0.96 g lecithin (Lipoid S 100) is dissolved in ethanol.The lecithin solution is added to the melt. The ethanol is evaporated ata temperature of 85° C. 160 mg sodium glycocholate, 0.9 g glycerol and 4mg thiomersal is dissolved in 35 ml bidistilled water. The solution isheated to 85° C. and added to the melt. A crude dispersion is preparedby ultra-turrax vortexing for approximately 2 minutes. The crudedispersion is transferred to a high-pressure homogenizer (APV GaulinMicron Lab 40) thermostatized at approximately 90° C. and passed 10times through the homogenizer at a pressure of 800 bar. The homogenizeddispersion is allowed to stand at room temperature to cool off.

Example 17

Preparation of tripalmitate SLPs without ultrasonication (method B).

In a thermostatized vial 4.0 g tripalmitate (Dynasan 116, Huls AG) ismelted at 85° C. 0.96 g lecithin (Lipoid S 100) is added to the melt.The mixture is shaken until the lecithin is completely dispersed in themelt and the dispersion appears isotropic. 160 mg sodium glycocholate,0.9 g glycerol and 4 mg thiomersal is dissolved in 35 ml bidistilledwater. The solution is heated to 85° C. and is added to the melt. Acrude dispersion is prepared by ultra-turrax vortexing for approximately2 minutes. The crude dispersion is transferred to a high-pressurehomogenizer (APV Gaulin Micron Lab 40) thermostatized at approximately90° C. and passed 10 times through the homogenizer at a pressure of 800bar. The homogenized dispersion is allowed to stand at room temperatureto cool off.

Example 18

Preparation of tripalmitate SLPs by dispersing phospholipids in theaqueous phase.

In a thermostatized vial 4.0 g tripalmitate (Dynasan 116) is melted at80° C. 0.96 g phospholipids (Lipoid S 100) is dispersed in 35 ml of anaqueous solution of 160 mg sodium glycocholate, 0.9 g glycerol and 4 mgthiomersal by stirring for approximately one hour. The phospholipiddispersion is heated to 80° C. and added to the tripalmitate melt. Acrude dispersion is prepared by probe sonication for approximately 2minutes. The crude dispersion is transferred to a high-pressurehomogenizer (APV Gaulin Micron Lab 40) thermostatized at approximately90° C. and passed 10 times through the homogenizer at a pressure of 800bar. The homogenized dispersion is allowed to stand at room temperatureto cool off.

Example 19

Preparation of tripalmitate SLPs stabilized by a highly mobilesurfactant.

In a thermostatized vial 5.0 g tripalmitate is melted at 80° C. 600 mgsodium glycocholate is dissolved in 44.4 g bidistilled water containing1.13 g glycerol and 0.01% thiomersal. The aqueous solution is heated to80° C. and added to the melt. A crude dispersion is prepared bysonication for approximately 5 minutes. The crude dispersion istransferred to a thermostatized high-pressure homogenizer (APV GaulinMicron Lab 40) and passed 8 times through the homogenizer at a pressureof 800 bar. The dispersion is allowed to stand at room temperature tocool off.

The mean particle size (by number) of the SLP dispersion afterpreparation is 96.8 nm determined by PCS. The size distribution isnarrow and monomodal.

This example demonstrates that it is possible to prepare smallhomogenously sized SLPs by the use of one surfactant only, such as thebile salt sodium glycocholate, provided the surfactant is highly mobileand constitutes a reservoir of stabilizer in the aqueous phase in orderto provide for the stabilization of newly created surfaces duringrecrystallization of the SLP matrix.

Example 20

Long-term stability of different SLP dispersions.

Several different SLP dispersions are prepared according to the methoddescribed in Example 2. All dispersions contain 2.25% glycerol asisotonicity agent and 0.01% thiomersal as a preservative. The long-termstability of the dispersions is judged from repeated size measurements(by PCS) over a period of 18 months. The dispersions are stored atrefrigeration temperatures. For comparison a soy bean oil emulsionsystem is included. The composition of the dispersions and their meanparticle sizes during storage are summarized in Table 4.

                  TABLE 4                                                         ______________________________________                                                            Mean particle size (nm)                                   Composition (w %)   after                                                     Matrix      Pl     SGC      Preparation                                                                           18 months                                 ______________________________________                                        10% TP      1.2%   0.4%     125.9   121.6                                     10% TP      2.4%   0.4%     104.5   111.2                                     10% TP      2.4%   0.4%     103.6   104.7                                     9.5% TP.sup.1)                                                                0.5% GMS    2.4%   0.4%     102.4   102.4                                     10% SO      2.4%   0.4%     129.6   139.9                                     ______________________________________                                         Abbreviations:                                                                PL = phospholipid (Lipoid S 100); SGC = sodium glycocholate; TP =             tripalmitate; GMS = glycerol monostearate; SO = soy bean oil.                 .sup.1) The SLP dispersion contains 5% (related to fat phase) of the          cardioprotective drug ubidecarenone.                                     

It is shown that the mean particle size of the dispersions remainspractically unchanged during storage for 18 months. Thus, the resultsdemonstrate that drug-free and drug-loaded SLP dispersions exhibit along-term stability similar to that of lipid emulsions.

Example 21

Sterile filtration of tripalmitate SLPs.

40 ml of a crude SLP dispersion composed of 3% tripalmitate (Dynasan116, Huls AG), 1.5% tyloxapol, 1% lecithin (Lipoid S 100), 2.25%glycerol and 0.01% thiomersal in bidistilled water to 100% is preparedaccording to the method described in Example 6. The crude dispersion ispassed 5 times through a thermostatized homogenizer (APV Micron Lab 40)at a pressure of 1200 bar. Half the volume of the batch is allowed tostand at room temperature to cool off, whereas the rest is filteredthrough a sterile syringe filter (Nalgene SFCA, 0.2 μm pore size) beforebeing cooled to the recrystallization temperature of the molten lipids.

The particle size distribution of both samples is determined by PCS. Themean particle size of the unfiltered sample is 56.7 nm and that of thesterile filtered SLP dispersion is 53.2 nm, i e both samples havepractically the same mean particle size.

Example 22

Sterilization of tripalmitate SLPs by autoclaving.

40 ml of a crude SLP dispersion composed of 3% tripalmitate (Dynasan116, Huls AG), 1.8% lecithin (Lipoid S 100), 0.6% sodium glycocholate,2.25% glycerol and 0.01% thiomersal in bidistilled water to 100% isprepared according to the method described in Example 2. The crudedispersion is passed 10 times through a thermostatized homogenizer (APVMicron Lab 40) at a pressure of 1200 bar.

Before being cooled to the recrystallization temperature of the moltenlipids the SLP dispersion is filled into an injection vial andsterilized by autoclaving at 121° C./2 atm for 45 minutes. Theautoclaved dispersion is allowed to stand at room temperature to cooloff. It shows no signs of aggregation or phase separation and has a meanparticle size of 65.9 nm determined by PCS.

Example 23

Lyophilization of SLPs.

In a thermostatized vial 3.5 g tripalmitate (Dynasan 116, Huls AG) ismelted at 75° C., and 1.05 g lecithin (Lipoid S 100, Lipoid KG) isdispersed in the melt by probe sonication. 1.58 g tyloxapol, 14 gsucrose and 7 mg thiomersal is dissolved in 50 ml bistilled water heatedto 75° C. and the aqueous phase is added to the lipid melt. A crudedispersion is prepared by probe sonication and then passed 5 timesthrough a thermostated high-pressure homogenizer (APV Gaulin Micron Lab40) at a pressure of 900 bar. The homogenized dispersion is passedthrough a 0.2 μm sterile filter.

For lyophilization the dispersion is filled into plane-bottom vialswhich are immersed in liquid nitrogen for 1 minute and transferred tothe freeze-drying chamber. Samples are freeze-dried for 36 hours undervacuum at a recipient temperature of -40° C.

Freeze-drying yields easily redispersible fine powders. The particlesize of the SLP dispersions is determined by PCS prior to lyophilizationand after reconstitution of the lyophilized powders with bistilledwater. The mean particle size prior to lyophilization is 79 nm and thatof the reconstituted dispersion is 87 nm, i e there is practically nochange in mean particle size after lyophilization.

Example 24

Surface modification by adsorption of polymers.

In a thermostalized vial 4.0 g tripalmitate (Dynasan 116, Huls AG) ismelted at 80° C. and 1.6 g soy bean lecithin (Lipoid S 100) is dispersedin the melt by probe sonication. 35.25 g of an aqueous solution of 0.01%thiomersal is heated to 80° C. and added to the melt. A crude dispersionis produced by probe sonication which is then passed five times througha high-pressure homogenizer at a pressure of 1200 bar. The dispersion isfiltered through a 0.2 μm syringe filter. The batch is divided into 3parts of equal volumes. One part is diluted with the same amount ofwater and stored at 90° C. to prevent gelation of the phospholipidstabilized dispersion on cooling down below the recrystallizationtemperature. The other two parts of the batch are incubated overnightwith equal volumes of 6% (w/w) poloxamer 407 (Pluronic F127, BASF) and6% (w/w) poloxamine 908 (Tetronic 908, BASF) solution, respectively, insuch a way that the polymer solution is added to the SLP dispersionprior to being cooled below the recrystallization temperature of SLPs toprovide for the immediate availability of polymer molecules as soon asnew surfaces are created due to recrystallization. Both polymers havebeen described in literature to modify the biodistribution ofintravenously administered colloidal particles.

The modification of the surface properties of tripalmitate SLPs isdemonstrated by differences in the zetapotential. Zetapotentials weredetermined by laser Doppler anemometry in a microelectrophoresis cell(Malvern Zetasizer 3). The results are summarized in Table 5.

                  TABLE 5                                                         ______________________________________                                        Zetapotential of surface modified SLPs.                                       Composition  % (w/w)!                                                                             Zetapotential                                             TP     PL          Polymer   mV!                                              ______________________________________                                        5%     2%          --       -29.6                                             5%     2%          3% F127  -1.9                                              5%     2%          3% T908  -2.9                                              ______________________________________                                         Abbreviations:                                                                TP = tripalmitate, PL = phospholipids, F127 = PLURONIC F127, T908 =           Tetronic 908.                                                            

The incubation of SLPs with block copolymers of the poloxamer andpoloxamine type results in a decrease of the zetapotential. Due to theadsorption of the polymers the surfaces become more hydrophobic. Thehydrophobicity of the surface is described to be one of the factorsgoverning the RES (reticuloendothelial system) activity and thebiodistribution of colloidal particles.

Example 25

Preparation of SLPs loaded with the cardio-protective drugubidecarenone.

Three different types of SLPs containing the cardio-protective drugubidecarenone are prepared. The SLPs are composed as summarized in table6. All dispersions contain 2.25% glycerol and 0.01% thiomersal.

Batch 1 and 2 are prepared by dispersing lecithin in the molten matrixconstituent as described before. In this melt ubidecarenone isdissolved. After addition of the aqueous phase containing sodiumglycocholate, glycerol and thiomersal a crude dispersion is prepared byprobe sonication. It is transferred to a thermostatized homogenizer (APVMicron Lab 40) and passed through the homogenizer ten times at apressure of 800 bar. The dispersions are allowed to stand at roomtemperature to cool off.

Batch 3 is prepared by dispersing lecithin in the molten matrixconstituent as described before. In this melt ubidecarenone isdissolved. After addition of the aqueous phase containing tyloxapol,glycerol and thiomersal, a crude dispersion is prepared by probesonication. It is transferred to a thermostatized homogenizer (APVMicron Lab 40) and passed 5 times through the homogenizer at a pressureof 1200 bar. The dispersion is allowed to stand at room temperature tocool off.

                  TABLE 6                                                         ______________________________________                                        Ubidecarenone-loaded SLPs.                                                    Batch no                                                                              Composition          Mean particle size                               ______________________________________                                        1       10% TP, 2.4% PL, 0.4% SGC, 1% Ubi                                                                  80.2 nm                                          2       10% HF, 1.2% PL, 0.4% SGC, 1% Ubi                                                                  78.9 nm                                          3       3% TP, 1.5% Tyl, 1% PL, 0.2% Ubi                                                                   46.8 nm                                          ______________________________________                                         Abbreviations:                                                                TP = tripalmitate, PL = phospholipids, SGC = sodium glycocholate, Ubi =       Ubidecarenone, HF = hard fat (Witepsol W35), Tyl = Tyloxapol.            

Example 26

Preparation of SLPs loaded with oxazepam.

In a thermostatized vial 7.0 g tripalmitate is melted at 80° C. 1.68 glecithin and 140 mg oxazepam is dispersed in the melt by probesonication. 60 ml of heated aqueous phase containing 280 mg sodiumglycocholate, 1.58 g glycerol and 7 mg thiomersal is added to the meltand a crude dispersion is prepared by probe sonication. The crudedispersion is homogenized by passing 10 times through a thermostatizedhigh-pressure homogenizer at a pressure of 800 bar. The dispersion isallowed to stand at room temperature to cool off. The dispersion ofoxazepam loaded SLPs has a mean particle size of 122.7 nm afterpreparation.

Example 27

Preparation and long-term stability of SLPs loaded with diazepam.

In a thermostatized vial 4.0 g tripalmitate is melted at 80° C. 0.96 glecithin and 120 mg diazepam is dispersed in the melt by probesonication. 35 ml of heated aqueous phase containing 160 mg sodiumglycocholate, 0.9 g glycerol and 4 mg thiomersal is added to the meltand a crude dispersion is prepared by probe sonication. The crudedispersion is homogenized by passing 10 times through a thermostatizedhigh-pressure homogenizer at a pressure of 800 bar. The dispersion isallowed to stand at room temperature to cool off.

The dispersion of diazepam-loaded SLPs has a mean particle size afterpreparation of 104.6 nm. After 12 months of storage the mean particlesize determined by PCS is 113.9 nm. Precipitation of drug substanceduring storage is not detected macroscopically. Investigations of thedispersion by polarized light microscopy over the monitored period of 12months do not reveal the presence of drug crystals.

Example 28

Preparation of SLPs loaded with lidocaine.

In a thermostatized vial 4.0 g tripalmitate is melted at 80° C. 0.96 glecithin and 60 mg lidocaine is dispersed in the melt by probesonication. 35 ml of heated aqueous phase containing 320 mg sodiumglycocholate, 0.9 g glycerol and 4 mg thiomersal is added to the meltand a crude dispersion is prepared by probe sonication. The crudedispersion is homogenized by passing 10 times through a thermostatizedhigh-pressure homogenizer at a pressure of 800 bar. The dispersion isallowed to stand at room temperature to cool off.

The dispersion of lidocaine loaded SLPs has a mean particle size afterpreparation of 90.4 nm.

Example 29

Preparation and long-term stability of SLPs loaded with prednisolone.

In a thermostatized vial 4.0 g tripalmitate is melted at 80° C. 0.48 glecithin and 80 mg prednisolone is dispersed in the melt by probesonication. 36 ml of heated aqueous phase containing 160 mg sodiumglycocholate, 0.9 g glycerol and 4 mg thiomersal is added to the meltand a crude dispersion is prepared by probe sonication. The crudedispersion is homogenized by passing 10 times through a thermostatizedhigh-pressure homogenizer at a pressure of 800 bar. The dispersion isallowed to stand at room temperature to cool off.

The dispersion of prednisolone-loaded SLPs has a mean particle sizeafter preparation of 118.3 nm. After 12 months of storage the meanparticle size determined by PCS is 124.2 nm. Precipitation of drugsubstance during storage is not detected macroscopically. Investigationsof the dispersion by polarized light microscopy over the monitoredperiod of 12 months do not reveal the presence of drug crystals.

Example 30

Preparation of SLPs loaded with cortisone.

Four different types of SLPs containing cortisone are prepared. The SLPsare composed as summarized in table 7. All dispersions contain 2.25%glycerol and 0.01 thiomersal.

Batch 1 and 2 are prepared by dispersing lecithin in the molten matrixconstituent as described before. In this melt cortisone is dissolved.After addition of the aqueous phase containing sodium glycocholate,glycerol and thiomersal, a crude dispersion is prepared by probesonication. It is transferred to a thermostatized homogenizer (APVMicron Lab 40) and passed 10 times through the homogenizer. Thedispersions are allowed to stand at room temperature to cool off.

Batch 3 is prepared by dispersing lecithin in the molten matrixconstituent as described before. In this melt cortisone is dissolved.After addition of the aqueous phase containing poloxamer (Pluronic F68),glycerol and thiomersal, a crude dispersion is prepared by probesonication. It is transferred to a thermostatized homogenizer (APVMicron Lab 40) and passed 5 times through the homogenizer at a pressureof 1200 bar. The dispersion is allowed to stand at room temperature tocool off.

Batch 4 is prepared by dispersing lecithin in the molten matrixconstituent as described before. In this melt cortisone is dissolved.After addition of the aqueous phase containing tyloxapol, glycerol andthiomersal a crude dispersion is prepared by probe sonication. It istransferred to a thermostatized homogenizer (APV Micron Lab 40) andpassed through the homogenizer five times at a pressure of 1200 bar. Thedispersion is allowed to stand at room temperature to cool off.

                  TABLE 7                                                         ______________________________________                                        Cortisone-loaded tripalmitate SLPs.                                           Batch no                                                                             Composition           Mean particle size                               ______________________________________                                        1      10% TP, 1.2% PL, 0.4% SGC, 0.2% Cort                                                                124.2 nm                                         2      3% TP, 1.8% PL, 0.6% SGC, 0.3% Cort                                                                 67.3 nm                                          3      10% TP, 4.5% Plu, 3% PL, 0.1% Cort                                                                  70.5 nm                                          4      3% TP, 1.5% Tyl, 1% PL, 0.1% Cort                                                                   48.5 nm                                          ______________________________________                                         Abbreviations:                                                                TP = tripalmitate, PL = phospholipids, SGC = sodium glycocholate, Cort =      Cortisone, Plu = Pluronic F68, Tyl = Tyloxapol.                          

Example 31

Tripalmitate SLPs loaded with retinol (vitamin A).

In a thermostatized vial 1.0 g tripalmitate (Dynasan 116, Huls AG) ismelted at 80° C. 60 mg retinol (vitamin A-alcohol >99%, Fluka) isdissolved in the melt. 300 mg soy bean lecithin (Lipoid S 100) isdispersed in the melt by probe sonication until the dispersion appearsoptically clear. 450 mg poloxamer 407 (Pluronic™ F127, BASF) isdissolved in 29.0 g bidistilled water. The aqueous phase is heated to80° C. and added to the melt. A fine dispersion is prepared by probesonication for 20 minutes. The dispersion is filtered through a 0.2 μmsyringe filter to remove metal shed from the ultrasound probe. Thedispersion is allowed to stand at room temperature to cool off.

The mean particle size by number after preparation of vitamin A-loadedtripalmitate SLPs is 98.5 nm determined by PCS.

Example 32

Tripalmitate SLPs loaded with phytylmenadione (vitamin K₃).

In a thermostatized vial 1.0 g tripalmitate (Dynasan 116, Huls AG) ismelted at 80° C. 60 mg phytylmenadione (vitamin K₃, Sigma) and 300 mgsoy bean lecithin (Lipoid S 100) is dispersed in the melt by probesonication until the dispersion appears optically clear. 450 mgpoloxamer 407 (Pluronic™ F127, BASF) is dissolved in 28.7 g bidistilledwater. The aqueous phase is heated to 80° C. and added to the melt. Afine dispersion is prepared by probe sonication for 20 minutes. Thedispersion is filtered through a 0.2 μm syringe filter to remove metalshed from the ultrasound probe. The dispersion is allowed to stand atroom temperature to cool off. The mean particle size by number afterpreparation of vitamin K₃ -loaded tripalmitate SLPs is 86.8 nmdetermined by PCS.

Example 33

Preparation of tripalmitate SLPs loaded with estramustine.

In a thermostatized vial 7.0 g tripalmitate (Dynasan 116, Huls AG) ismelted at 80° C. In the melt 1.68 g soy bean lecithin (Lipoid S 100) isdispersed by probe sonication until the dispersion appears opticallyclear. 40 mg estramustine is dissolved in the tripalmitate/lecithindispersion. 0.42 g sodium glycocholate and 1.58 g glycerol is dissolvedin 60 g bidistilled water. The aqueous phase is heated to 80° C. andadded to the melt. A crude emulsion is prepared by probe sonication forapproximately 2 minutes. The crude emulsion is transferred to athermostatized high-pressure homogenizer (APV Gaulin Micron Lab 40) andpassed 10 times through the homogenizer at a pressure of 800 bar. Thedispersion is allowed to stand at room temperature to cool off.

Example 34

Physical state of different SLPs at body temperature.

Two batches of SLPs from different matrix constituents are preparedaccording to the method described in Example 2. Batch 1 is composed of10% tripalmitate, 0.5% ubidecarenone, 1.2% soy bean lecithin (Lipoid S100, Lipoid KG), 0.4% sodium glycocholate, 2.25% glycerol, 0.01%thiomersal and bidistilled water to 100% (by weight). Batch 2 iscomposed of 10% hard fat (Witepsol™ W35, Huls AG), 0.5% ubidecarenone,1.2% soy bean lecithin (Lipoid S 100, Lipoid KG), 0.4% sodiumglycocholate, 2.25% glycerol, 0.01% thiomersal and bidistilled water to100% (by weight).

The physical state of the matrix constituents is determined bysynchrotron radiation X-ray diffraction at 20° C. and at 38° C. Thesamples are placed in thermostatized sample holders. The diffractionpatterns are recorded for 180 seconds each. FIG. 15a demonstrates thatat room temperature (20° C.) both batches of SLPs are crystalline. Thespacings correspond to the crystalline polymorphs. At body temperature(38° C.) the tripalmitate-SLPs are still crystalline whereas noreflections can be detected for the hard fat SLPs, i e they areamorphous and molten (FIG. 15b). The different physical states of theseSLPs at body temperature give rise to a different biopharmaceuticalbehaviour with respect to the release of incorporated drugs or bioactiveagents. SLPs molten at body temperature display basically the releasecharacteristics typical of conventional lipid emulsions. Due to the freediffusion of drug molecules in the liquid lipid the drug can be releasedfrom the vehicle relatively fast. In contrast, SLPs which are solid atbody temperature give rise to sustained release of incorporated drugs.Since the drug molecules are immobilized in the solid matrix drugrelease is not diffusion-controlled but depends on the degradation ofthe solid lipid matrix in the body and is therefore delayed.

Example 35

Preparation of PBAs from miconazole.

In a thermostatized vial 0.4 g miconazole is melted at 90° C. 0.24 glecithin (Lipoid S 100) is dispersed in the melt by probe sonicationuntil the dispersion appears optically clear. 0.9 g glycerol, 80 mgsodium glycocholate and 4 mg thiomersal is dissolved in 38.5 mlbidistilled water and heated to 90° C. The aqueous phase is added to themiconazole/lecithin melt and a crude dispersion is produced by probesonication for 5 minutes. The crude dispersion is transferred to athermostatized high-pressure homogenizer (APV Gaulin Micron Lab 40) andpassed 10 times through the homogenizer at a pressure of 800 bar. ThePBA dispersion is allowed to stand at room temperature to cool off.

On cooling the molten miconazole recrystallizes and forms a suspensionof miconazole microparticles. The mean particle size (by volume) ofmiconazole PBAs is 21.8 μm determined by laser diffractometry. Thesediment of miconazole PBAs is easily redispersible by slight agitation.

Example 36

Preparation of PBAs from ibuprofen.

In a thermostatized vial 1.2 g ibuprofen is melted at 85° C. 0.72 glecithin (Lipoid S 100) is dispersed in the melt by probe sonicationuntil the dispersion appears optically clear. 0.9 g glycerol, 240 mgsodium glycocholate and 4 mg thiomersal is dissolved in 37 mlbidistilled water and heated to 85° C. The aqueous phase is added to theibuprofen/lecithin melt and a crude dispersion is produced by probesonication for 5 minutes. The crude dispersion is transferred to athermostatized high-pressure homogenizer (APV Gaulin Micron Lab 40) andpassed 6 times through the homogenizer at a pressure of 800 bar. The PBAdispersion is allowed to stand at room temperature to cool off.

On cooling the molten ibuprofen recrystallizes and forms a suspension ofibuprofen microparticles. The mean particle size (by volume) ofibuprofen PBAs is 61.4 μm determined by laser diffractometry. Thesediment of ibuprofen PBAs is easily redispersible by slight agitation.

Example 37

Dissolution speed of ibuprofen PBAs.

The dissolution speed of ibuprofen PBAs of Example 36 is measured in alaser diffractometer (Malvern Mastersizer MS20) by monitoring the decayof the so-called obscuration over a period of 10 minutes. Theobscuration is a measure of the reduced intensity of unscattered laserlight by a sample and is related to the concentration of particles inthe laser beam. In parallel the particle size can be measured. Formeasurement the ibuprofen PBA sample is diluted with water and dispersedby magnetic stirring in a measuring cell placed in the laser beam line.FIG. 16 presents the decay of obscuration and particle size of a sampleof ibuprofen PBAs. Within 10 minutes the obscuration has decayed tozero, i.e. there is no detectable amount of particles hinting at thecomplete dissolution of the PBAS. The dissolution of the untreated rawsubstance ibuprofen cannot be measured by this technique since thesubstance is only poorly wettable in water.

Example 38

Preparation of PBAs from lidocaine.

In a thermostatized vial 1.2 g lidocaine is melted at 80° C. 1.2 gtyloxapol is dissolved in 37.6 ml bidistilled water and heated to 80° C.The aqueous phase is added to the lidocaine melt and a crude dispersionis produced by probe sonication for 2 minutes. The crude dispersion istransferred to a thermostatized high-pressure homogenizer (APV GaulinMicron Lab 40) and passed 5 times through the homogenizer at a pressureof 1200 bar. The PBA dispersion is allowed to stand at room temperatureto cool off.

On cooling the molten lidocaine recrystallizes into fine needles andforms a suspension of lidocaine microparticles. FIG. 17 shows apolarized microscopic picture of the suspended lidocaine needles. Theparticle shape of the raw material lidocaine-base (Synopharm) isdifferent from that of lidocaine PBAs as demonstrated by the polarizedmicroscopic picture of FIG. 18.

The mean particle size (by volume) of lidocaine PBAs is 174.2 μmdetermined by laser diffractometry. The maximum detected particle sizeis 400 μm. The sediment of lidocaine PBAs is easily redispersible byslight agitation. The addition of water to PBAs leads to the rapiddissolution of the particles. In contrast the raw material lidocaine isonly sparingly soluble in water and the dissolution speed is muchslower. The high dissolution speed of lidocaine PBAs is a consequence ofthe modified surface properties and the finely dispersed state of theparticles. Due to the rapid dissolution a determination of thedissolution speed according to the method described in Example 37 is notpossible.

Example 39

Preparation of PBAs from cholecalciferol (vitamin D₃).

In a thermostatized vial 0.8 g cholecalciferol is melted at 95° C. 120mg soy bean lecithin (Lipoid S 100) is dispersed in the melt by probesonication until the dispersion appears optically clear. 40 mg sodiumglycocholate and 0.9 g glycerol is dissolved in 37.92 ml bidistilledwater and heated to 95° C. The aqueous phase is added to thecholecalciferol/lecithin dispersion and a crude dispersion is producedby probe sonication for 5 minutes. The crude dispersion is transferredto a thermostatized high-pressure homogenizer (APV Gaulin Micron Lab 40)and passed 8 times through the homogenizer at a pressure of 1200 bar.The PBA dispersion is allowed to stand at room temperature to cool off.

The mean particle size after preparation by number of cholecalciferolPBAs is 325.1 nm determined by PCS.

Example 40

Preparation of PBAs from estramustine.

In a thermostatized vial 2 g estramustine is melted at 105° C. In themelt 0.8 g soy bean lecithin (Lipoid S 100) is dispersed by probesonication until the dispersion appears optically clear. 0.2 g sodiumglycocholate and 0.9 g glycerol is dissolved in 36.1 g bidistilledwater. The aqueous phase is heated to 95° C. and added to the melt. Acrude emulsion is prepared by probe sonication for approximately 5minutes. The crude emulsion is transferred to a thermostatizedhigh-pressure homogenizer (APV Gaulin Micron Lab 40) and passed 5 timesthrough the homogenizer at a pressure of 1200 bar. The dispersion isallowed to stand at room temperature to cool off.

We claim:
 1. A process for transferring insoluble or sparingly watersoluble agents which are solid at room temperature into suspensions,characterized in that said suspensions is/are colloidal, long-termstable, of narrow size distribution and allow(s) high particleconcentration(s) and that the following steps are carried outa. thesolid agent or a mixture of solid agents is melted, b. a dispersionmedium is heated to approximately the same temperature as the moltensolid agent or the mixture of molten solid agents, c. one or more highlymobile water-soluble or dispersible stabilizers is/are added to thedispersion medium in such a way that the amount of highly mobilestabilizers is, after emulsification, sufficient to stabilize newlycreated surfaces during recrystallization, optionally, one or morelipid-soluble or dispersible stabilizers is are added additionally tothe melted agent or mixture of agents, d. the melted agent or mixture ofagents and the dispersion medium is/are premixed to a crude dispersionand subsequently homogenized by high-pressure homogenization,micro-fluidization and/or ultrasonication, e. the homogenized dispersionis allowed to cool until solid particles are formed by recrystallizationof the dispersed agents.
 2. A process according to claim 1,characterized in that the homogenized dispersion is passed through afilter prior to cooling below the recrystallization temperature toremove particulate contaminations in such a way that the filter poresize is chosen large enough not to retain the particles of emulsified,molten agents.
 3. A process according to claim 1, characterized in thatthe solid agent or mixture of solid agents is/are a lipid/lipids havingmelting points between approximately 30° C. and 120° C. and areconstituted of mono-, di- and triglycerides of long chain fatty acids;hydrogenated vegetable oils; fatty acids and their esters; fattyalcohols and their esters and ethers; natural or synthetic waxes; waxalcohols and their esters; sterols; hard paraffins; or mixtures of theabove mentioned lipids.
 4. A process according to claim 1, characterizedin that the solid agent or mixture of solid agents is/are a bioactiveagent/bioactive agents or drug/drugs showing a low bioavailabilityand/or being badly absorbed from the instestinum and having meltingpoints below about 100° C. or the melting points of which can bedecreased to below about 100° C. by addition of adjuvants, suchbioactive agent/bioactive agents or drug/drugs being anesthetics andnarcotics, anticholinergics, antidepressives, psychostimulants andneuroleptics, antiepileptics, antimycotics, antiphlogistics,bronchodilators, cardiovascular drugs, cytostatics, hyperemic drugs,lipid reducers, spasmolytics, testosterone derivatives, tranquilizers,virustatics, vitamin A derivatives, vitamin E derivatives, menadione,cholecalciferol, insecticides, pesticides and/or herbicides.
 5. Aprocess according to claim 4, characterized in that the solid agent ormixture of solid agents is/are a bioactive agent/bioactive agents ordrug/drugs showing a low bioavailability and/or being badly absorbedfrom the instestinum and having melting points below about 100° C. orthe melting points of which can be decreased to below about 100° C. andbeing constituted of butanilicaine, fomocaine, isobutambene, lidocaine,risocaine, pseudococaine, prilocaine, tetracaine, trimecaine,tropacocaine, etomidate, metixen, profenamine, alimenazine, binedaline,perazine, chlorpromazine, fenpentadiol, fenanisol, mebenazine,methylphenidate, thioridazine, toloxaton, trimipramide, dimethadion,nicethamide, butoconazole, chlorphenesin, etisazole, exalamid,precilocine, miconazole, butibufen, ibuprofin, bamifylline, alprenolol,butobendine, clordiazole, hexobendine, nicofibrate, penbutolol,pirmenol, prenylamine, procaine amide, propatrylnitrate, suloctidil,toliprolol, xidbendol, viquidile, asperline, chlorambucile, mitotane,estramustine, taxol, penclomedine, trofosfamide, capsaicine,methylnicotinate, nicolclonate, oxprenolol, pirifibrate, simfibrate,thiadenol, aminopromazine, caronerine, difemerine, fencarbamide,tiropramide, moxaverine, testosterone enantate,testosterone-(4-methylpentanoate), azaperone, buramate, arildon,retinol, retinol acetate, retinol palmitate, tocopherol acetate,tocopherol succinate, tocopherol nicotinate, menadione, cholecalciferol,acephate, cyfluthrin, azinphosphomethyl, cypermethrine, substitutedphenyl thiophosphates, fenclophos, permethrine, piperonal, tetramethrineand/or trifluraline.
 6. A process according to claim 1, characterized inthat the surface characteristics of the particles are modified afterhomogenization in order to control the biodistribution of the particles.7. A process according to claim 1, characterized in that during coolingthe dispersion is agitated.
 8. A process according to claim 1,characterized in that the dispersion medium is a pharmacologicallyacceptable liquid not dissolving the agent or mixture of agents.
 9. Aprocess according to claim 8, characterized in that the dispersionmedium is a pharmacologically acceptable liquid not dissolving the agentor mixture of agents selected from the group consisting of water,ethanol, propylene glycol, dimethyl sulfoxide (DMSO),methyl-isobutyl-ketone and mixtures thereof.
 10. A process according toclaim 1, characterized in that the stabilizer or stabilizers areamphiphatic compounds, physiological bile salts, saturated andunsaturated fatty acids or fatty alcohols, ethoxylated fatty acids orfatty alcohols and their esters and ethers, alkylaryl-polyetheralcohols, esters and ethers of sugars or sugar alcohols with fatty acidsor fatty alcohols, acetylated or ethoxylated mono- and diglycerides,synthetic biodegradable polymers, ethoxylated sorbitanesters orsorbitanethers, amino acids, polypeptides and proteins or a combinationof two or more of the above mentioned stabilizers.
 11. A processaccording to claim 10, characterized in that the stabilizer orstabilizers are selected from the group consisting of ionic andnon-ionic surfactants, naturally occurring and synthetic phospholipids,their hydrogenated derivatives and mixtures thereof, sphingolipids andglycosphingolipids, sodium cholate, sodium dehydrocholate, sodiumdeoxycholate, sodium glycocholate, sodium taurocholate, tyloxapol, blockco-polymers of polyoxyethylene and polyoxypropyleneoxide, gelatin andalbumin and a combination of two or more of the above mentionedstabilizers.
 12. A process according to claim 1, characterized in thatthe stabilizer or stabilizers is/are a combination of phospholipids andbile salts.
 13. A process according to claim 12, characterized in thatthe molar ratio of phospholipids to bile salts is about 2:1 or above.14. A process according to claim 12, characterized in that thedispersion medium contains isotonicity agents and/or cryoprotectants.15. A process according to claim 14, characterized in that theisotonicity agent is glycerol and the cryoprotectant is a sugar or sugaralcohol.
 16. A process according to claim 1, characterized in that thestabilizer or stabilizers is/are a combination of phospholipids andsodium glycocholate in a molar ratio between about 2:1 and about 4:1.17. A process according to claim 1, characterized in that the dispersionmedium contains one or more of the following additives: water-soluble ordispersable stabilizers; isotonicity agents; cryoprotectants;electrolytes; buffers; antiflocculants; and preservatives.
 18. A processaccording to claim 17, characterized in that the dispersion mediumcontains one or more of the following additives: water-soluble ordispersable stabilizers, glycerol, xylitol, sucrose, glucose, maltose,trehalose, sodium citrate, sodium pyrophosphate and sodiumdodecylsulfate.
 19. A process according to claim 1, characterized inthat the dispersion is sterilized prior to cooling down the dispersionbelow the recrystallization temperature of the molten lipids.
 20. Aprocess according to claim 1, characterized in that in a subsequent stepthe dispersion medium is reduced in volume, yielding liquid-freeparticles which can be reconstituted prior to use.
 21. A suspension ofcolloidal solid lipid particles (SLPs) manufactured according to claim1, characterized in that the SLPs are lipids having melting pointsbetween approximately 30° C. and 120° C. and are constituted of mono-di- and triglycerides of long chain fatty acids; hydrogenated vegetableoils; fatty acids and their esters; fatty alcohols and their esters andethers; natural or synthetic waxes; wax alcohols and their esters;sterols; hard paraffins; or mixtures of the above-mentioned lipids, andfurther characterized in that the particles are stabilized by acombination of phospholipids and bile salts.
 22. A suspension ofcolloidal solid lipid particles (SLPs) according to claim 21,characterized in that the molar ratio of phospholipids to bile salts isabout 2:1 or above.
 23. A suspension of colloidal particles (SLPs)according to claim 21, characterized in that the dispersion mediumcontains isotonicity agents and/or cryoprotectants.
 24. A suspension ofcolloidal particles (SLPs) according to claim 23, characterized in thatthe dispersion medium contains glycerol and/or sugars or sugar alcohols.25. A suspension of colloidal solid lipid particles (SLPs) according toclaim 21, characterized in that the SLPs are of a non-α-like crystallinemodification at a temperature below the melting temperature.
 26. Asuspension of colloidal solid lipid particles (SLPs) according to claim21, characterized in that the SLPs are of a non-spherical shape at atemperature below the melting temperature.
 27. A suspension of colloidalsolid lipid particles (SLPs) according to claim 21, characterized inthat the particles are of micron or submicron size, predominantly in thesize range from 20 to 500 nm.
 28. A suspension of colloidal solid lipidparticles (SLPs) according to claim 21, characterized in that into theSLPs are entrapped drugs or bioactive compounds which are poorlywater-soluble, show a low bioavailability, are badly absorbed from theintestinum and/or are rapidly degraded in a biological environment bychemical or enzymatical processes.
 29. A suspension of colloidal solidlipid particles (SLPs) according to claim 28, characterized in that theentrapped drugs are antibiotics, antihypertensives, antihypotensives,systemic antimycotics, antiphlogistics, antiviral agents,immunoglobulins, ACE inhibitors, betablockers, bronchodilators, calciumantagonists, cardiac glycosides, cephalosporins, cytostatics, hypnotics,psychotropic drugs, steroid hormones, vasodilators, cerebralvasodilators, and lipophilic vitamins and their derivatives.
 30. Asuspension of colloidal solid lipid particles (SLPs) according to claim29, characterized in that the entrapped drugs are selected from thegroup consisting of fosfomycin, fosmidomycin, rifapentin, minoxidil,dihydroergotoxine, endralazine, dihydroergotamine, ketoconazole,griseofulvin, indomethacin, diclofenac, ibuprofen, ketoprofen,pirprofen, aciclovir, vidarabin, captopril, enalapril, propranolol,atenolol, metoprolol, pindolol, oxprenolol, labetalol,ipratropiumbromide, sobrerol, diltiazem, flunarizin, verapamil,nifedipin, nimodipin, nitrendipin, digitoxin, digoxin, methyldigoxin,acetyldigoxin, nitrendipin, digitoxin, digoxin, methyldigoxin,acetyldigoxin, ceftizoxim, cefalexin, cefalotin, cefotaxim, chlormethin,cyclophosphamid, chlorambucil, cytarabin, vincristin, mitomycin C,doxorubicin, bleomycin, cisplatin, taxol, penclomedine, estramustine,flurazepam, nitrazepam and lorazepam, oxazepam, diazepam, bromazepam,cortisone, hydrocortisone, prednisone, prednisolone, dexamethasone,progesterone, pregnanolone, testosterone, testosteroneundecanoate,molsidomin, hydralazin, dihydralazin, ciclonicat, vincamin, and VitaminsA, D, E, K and their derivatives.
 31. Liquid-free particles manufacturedby removing the dispersion medium from a suspension according to claim21 by filtration, ultrafiltration or freeze-drying.
 32. A pharmaceuticalor medical composition which includes liquid-free particles as set forthin claim
 31. 33. In the therapeutic treatment in a living human oranimal body, the improvement comprising administering a suspensionaccording to claim 21, but not containing insecticides, pesticides orherbicides.
 34. A suspension according to claim 21 for use as amedicament.
 35. A pharmaceutical or medical composition which includes asuspension as set forth in claim
 21. 36. A suspension of colloidal solidlipid particles (SLPs) manufactured according to claim 1, characterizedin that the SLPs are lipids having melting points between approximately30° C. and 120° C. and are constituted of mono-, di- and triglyceridesof long chain fatty acids; hydrogenated vegetable oils; fatty acids andtheir esters; fatty alcohols and their esters and ethers; natural orsynthetic waxes; wax alcohols and their esters; sterols; hard paraffins;or mixtures of the above-mentioned lipids, and further characterized inthat the particles are stabilized by a combination of phospholipids andsodium glycocholate in a molar ratio between about 2:1 and about 4:1.